Secondary literature sources for Ald_Xan_dh_C

The following references were automatically generated.

Morais-Silva FO et al.
Genome sequence of the model sulfate reducer Desulfovibrio gigas: a comparative analysis within the Desulfovibrio genus.
Microbiologyopen. 2014; 3: 513-30
Display abstract
Desulfovibrio gigas is a model organism of sulfate-reducing bacteria of which energy metabolism and stress response have been extensively studied. The complete genomic context of this organism was however, not yet available. The sequencing of the D. gigas genome provides insights into the integrated network of energy conserving complexes and structures present in this bacterium. Comparison with genomes of other Desulfovibrio spp. reveals the presence of two different CRISPR/Cas systems in D. gigas. Phylogenetic analysis using conserved protein sequences (encoded by rpoB and gyrB) indicates two main groups of Desulfovibrio spp, being D. gigas more closely related to D. vulgaris and D. desulfuricans strains. Gene duplications were found such as those encoding fumarate reductase, formate dehydrogenase, and superoxide dismutase. Complexes not yet described within Desulfovibrio genus were identified: Mnh complex, a v-type ATP-synthase as well as genes encoding the MinCDE system that could be responsible for the larger size of D. gigas when compared to other members of the genus. A low number of hydrogenases and the absence of the codh/acs and pfl genes, both present in D. vulgaris strains, indicate that intermediate cycling mechanisms may contribute substantially less to the energy gain in D. gigas compared to other Desulfovibrio spp. This might be compensated by the presence of other unique genomic arrangements of complexes such as the Rnf and the Hdr/Flox, or by the presence of NAD(P)H related complexes, like the Nuo, NfnAB or Mnh.

Dong C, Yang J, Leimkuhler S, Kirk ML
Pyranopterin dithiolene distortions relevant to electron transfer in xanthine oxidase/dehydrogenase.
Inorg Chem. 2014; 53: 7077-9
Display abstract
The reducing substrates 4-thiolumazine and 2,4-dithiolumazine have been used to form Mo(IV)-product complexes with xanthine oxidase (XO) and xanthine dehydrogenase. These Mo(IV)-product complexes display an intense metal-to-ligand charge-transfer (MLCT) band in the near-infrared region of the spectrum. Optical pumping into this MLCT band yields resonance Raman spectra of the Mo site that are devoid of contributions from the highly absorbing FAD and 2Fe2S clusters in the protein. The resonance Raman spectra reveal in-plane bending modes of the bound product and low-frequency molybdenum dithiolene and pyranopterin dithiolene vibrational modes. This work provides keen insight into the role of the pyranopterin dithiolene in electron-transfer reactivity.

Pushie MJ, Cotelesage JJ, George GN
Molybdenum and tungsten oxygen transferases--and functional diversity within a common active site motif.
Metallomics. 2014; 6: 15-24
Display abstract
Molybdenum and tungsten are the only second and third-row transition elements with a known function in living organisms. The molybdenum and tungsten enzymes show common structural features, with the metal being bound by a pyranopterin-dithiolene cofactor called molybdopterin. They catalyze a variety of oxygen transferase reactions coupled with two-electron redox chemistry in which the metal cycles between the +6 and +4 oxidation states usually with water, either product or substrate, providing the oxygen. The functional roles filled by the molybdenum and tungsten enzymes are diverse; for example, they play essential roles in microbial respiration, in the uptake of nitrogen in green plants, and in human health. Together, the enzymes form a superfamily which is among the most prevalent known, being found in all kingdoms of life. This review discusses what is known of the active site structures and the mechanisms, together with some recent insights into the evolution of these important enzyme systems.

Kim-Shapiro DB, Gladwin MT
Mechanisms of nitrite bioactivation.
Nitric Oxide. 2014; 38: 58-68
Display abstract
It is now accepted that the anion nitrite, once considered an inert oxidation product of nitric oxide (NO), contributes to hypoxic vasodilation, physiological blood pressure control, and redox signaling. As such, its application in therapeutics is being actively tested in pre-clinical models and in human phase I-II clinical trials. Major pathways for nitrite bioactivation involve its reduction to NO by members of the hemoglobin or molybdopterin family of proteins, or catalyzed dysproportionation. These conversions occur preferentially under hypoxic and acidic conditions. A number of enzymatic systems reduce nitrite to NO and their activity and importance are defined by oxygen tension, specific organ system and allosteric and redox effectors. In this work, we review different proposed mechanisms of nitrite bioactivation, focusing on analysis of kinetics and experimental evidence for the relevance of each mechanism under different conditions.

Cao H, Pauff JM, Hille R
X-ray crystal structure of a xanthine oxidase complex with the flavonoid inhibitor quercetin.
J Nat Prod. 2014; 77: 1693-9
Display abstract
Xanthine oxidase catalyzes the sequential hydroxylation of hypoxanthine to uric acid via xanthine as intermediate. Deposition of crystals of the catalytic product uric acid or its monosodium salt in human joints with accompanying joint inflammation is the major cause of gout. Natural flavonoids are attractive leads for rational design of preventive and therapeutic xanthine oxidase inhibitors due to their beneficial antioxidant, anti-inflammatory, and antiproliferative activities in addition to their micromolar inhibitory activities toward xanthine oxidase. We determined the first complex X-ray structure of mammalian xanthine oxidase with the natural flavonoid inhibitor quercetin at 2.0 A resolution. The inhibitor adopts a single orientation with its benzopyran moiety sandwiched between Phe 914 and Phe 1009 and ring B pointing toward the solvent channel leading to the molybdenum active center. The favorable steric complementarity of the conjugated three-ring structure of quercetin with the active site and specific hydrogen-bonding interactions of exocyclic hydroxy groups with catalytically relevant residues Arg 880 and Glu 802 correlate well with a previously reported structure-activity relationship of flavonoid inhibitors of xanthine oxidase. The current complex provides a structural basis for the rational design of flavonoid-type inhibitors against xanthine oxidase useful for the treatment of hyperuricemia, gout, and inflammatory disease states.

Mendel RR
The molybdenum cofactor.
J Biol Chem. 2013; 288: 13165-72
Display abstract
The transition element molybdenum needs to be complexed by a special cofactor to gain catalytic activity. Molybdenum is bound to a unique pterin, thus forming the molybdenum cofactor (Moco), which, in different variants, is the active compound at the catalytic site of all molybdenum-containing enzymes in nature, except bacterial molybdenum nitrogenase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also require iron, ATP, and copper. After its synthesis, Moco is distributed, involving Moco-binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms.

Soriano EV et al.
Active-site models for complexes of quinolinate synthase with substrates and intermediates.
Acta Crystallogr D Biol Crystallogr. 2013; 69: 1685-96
Display abstract
Quinolinate synthase (QS) catalyzes the condensation of iminoaspartate and dihydroxyacetone phosphate to form quinolinate, the universal precursor for the de novo biosynthesis of nicotinamide adenine dinucleotide. QS has been difficult to characterize owing either to instability or lack of activity when it is overexpressed and purified. Here, the structure of QS from Pyrococcus furiosus has been determined at 2.8 A resolution. The structure is a homodimer consisting of three domains per protomer. Each domain shows the same topology with a four-stranded parallel beta-sheet flanked by four alpha-helices, suggesting that the domains are the result of gene triplication. Biochemical studies of QS indicate that the enzyme requires a [4Fe-4S] cluster, which is lacking in this crystal structure, for full activity. The organization of domains in the protomer is distinctly different from that of a monomeric structure of QS from P. horikoshii [Sakuraba et al. (2005), J. Biol. Chem. 280, 26645-26648]. The domain arrangement in P. furiosus QS may be related to protection of cysteine side chains, which are required to chelate the [4Fe-4S] cluster, prior to cluster assembly.

Maisel T, Joseph S, Mielke T, Burger J, Schwarzinger S, Meyer O
The CoxD protein, a novel AAA+ ATPase involved in metal cluster assembly: hydrolysis of nucleotide-triphosphates and oligomerization.
PLoS One. 2012; 7: 47424-47424
Display abstract
CoxD of the alpha-proteobacterium Oligotropha carboxidovorans is a membrane protein which is involved in the posttranslational biosynthesis of the [CuSMoO(2)] cluster in the active site of the enzyme CO dehydrogenase. The bacteria synthesize CoxD only in the presence of CO. Recombinant CoxD produced in E. coli K38 pGP1-2/pETMW2 appeared in inclusion bodies from where it was solubilized by urea and refolded by stepwise dilution. Circular dichroism spectroscopy revealed the presence of secondary structural elements in refolded CoxD. CoxD is a P-loop ATPase of the AAA-protein family. Refolded CoxD catalyzed the hydrolysis of MgATP yielding MgADP and inorganic phosphate at a 1ratio1ratio1 molar ratio. The reaction was inhibited by the slow hydrolysable MgATP-gamma-S. GTPase activity of CoxD did not exceed 2% of the ATPase activity. Employing different methods (non linear regression, Hanes and Woolf, Lineweaver-Burk), preparations of CoxD revealed a mean K(M) value of 0.69+/-0.14 mM ATP and an apparent V(max) value of 19.3+/-2.3 nmol ATP hydrolyzed min(-)(1) mg(-)(1). Sucrose density gradient centrifugation and gel filtration showed that refolded CoxD can exist in various multimeric states (2-mer, 4-mer or 6-mer), preferentially as hexamer or dimer. Within weeks the hexamer dissociates into the dimer, a process which can be reversed by MgATP or MgATP-gamma-S within hours. Only the hexamers and the dimers exhibited MgATPase activity. Transmission electron microscopy of negatively stained CoxD preparations revealed distinct particles within a size range of 10-16 nm, which further corroborates the oligomeric organization. The 3D structure of CoxD was modeled with the 3D structure of BchI from Rhodobacter capsulatus as template. It has the key elements of an AAA+ domain in the same arrangement and at same positions as in BchI and displays the characteristic inserts of the PS-II-insert clade. Possible functions of CoxD in [CuSMoO(2)] cluster assembly are discussed.

Mendel RR, Kruse T
Cell biology of molybdenum in plants and humans.
Biochim Biophys Acta. 2012; 1823: 1568-79
Display abstract
The transition element molybdenum (Mo) needs to be complexed by a special cofactor in order to gain catalytic activity. With the exception of bacterial Mo-nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor Moco, which in different variants is the active compound at the catalytic site of all other Mo-containing enzymes. In eukaryotes, the most prominent Mo-enzymes are nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and the mitochondrial amidoxime reductase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also requires iron, ATP and copper. After its synthesis, Moco is distributed to the apoproteins of Mo-enzymes by Moco-carrier/binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms. In humans, Moco deficiency is a severe inherited inborn error in metabolism resulting in severe neurodegeneration in newborns and causing early childhood death. This article is part of a Special Issue entitled: Cell Biology of Metals.

Kirk ML, Berhane A
Correlating C-H bond cleavage with molybdenum reduction in xanthine oxidase.
Chem Biodivers. 2012; 9: 1756-60
Display abstract
We have performed a computational study of substrate C-H bond activation in enzymes of the XO family. The C-H H-atom for all XO substrates studied is transferred to the terminal sulfido at the transition state with near neutral charge, and this is consistent with both Mo=S pi--> C-H sigma* and C-H sigma-->Mo=S pi* donor-acceptor interactions activating the C-H bond. A C-H bond scission and Mo reduction appear to be highly correlated along the reaction coordinate for all XO substrates studied, with Mo reduction being a continuous and exponential function of C-H bond breaking along the reaction coordinate.

Shi T, Xie J
Molybdenum enzymes and molybdenum cofactor in mycobacteria.
J Cell Biochem. 2011; 112: 2721-8
Display abstract
When intracelluar pathogens enter the host macrophages where in addition to oxidative and antibiotic mechanisms of antimicrobial activity, nutrients are deprived. Human pathogen Mycobacterium tuberculosis is one of macrophage parasitisms, which can replicate and persist for decades in dormancy state in virulent environments. It is very successful in escaping the killing mechanisms of macrophage. Molybdenum (Mo) enzymes involve in the global carbon, sulfur, and nitrogen cycles by catalyzing important redox reactions. There are several Mo enzymes in mycobacteria and they exert several important physiological functions, such as dormancy regulation, the metabolism of energy sources, and nitrogen source. Pterin-based Mo cofactor (Moco) is the common cofactor of the Mo enzymes in mycobacteria but the cofactor biosynthesis is nearly an untapped area. The present article discusses the physiological function of Mo enzymes and the structural feature of the genes coding for Moco biosynthesis enzymes in mycobacteria.

Cooley RB, Rhoads TW, Arp DJ, Karplus PA
A diiron protein autogenerates a valine-phenylalanine cross-link.
Science. 2011; 332: 929-929
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All known internal covalent cross-links in proteins involve functionalized groups having oxygen, nitrogen, or sulfur atoms present to facilitate their formation. Here, we report a carbon-carbon cross-link between two unfunctionalized side chains. This valine-phenyalanine cross-link, produced in an oxygen-dependent reaction, is generated by its own carboxylate-bridged diiron center and serves to stabilize the metallocenter. This finding opens the door to new types of posttranslational modifications, and it demonstrates new catalytic potential of diiron centers.

Vey JL, Drennan CL
Structural insights into radical generation by the radical SAM superfamily.
Chem Rev. 2011; 111: 2487-506

Drew SC, Reijerse E, Quentmeier A, Rother D, Friedrich CG, Lubitz W
Spectroscopic characterization of the molybdenum cofactor of the sulfane dehydrogenase SoxCD from Paracoccus pantotrophus.
Inorg Chem. 2011; 50: 409-11
Display abstract
The bacterial sulfane dehydrogenase SoxCD is a distantly related member of the sulfite oxidase (SO) enzyme family that is proposed to oxidize protein-bound sulfide (sulfane) of SoxY as part of a multienzyme mechanism of thiosulfate metabolism. This study characterized the molybdenum cofactor of SoxCD1, comprising the catalytic molybdopterin subunit SoxC and the truncated c-type cytochrome subunit SoxD1. Electron paramagnetic resonance spectroscopy of the Mo(V) intermediate generated by dithionite reduction revealed low- and high-pH species with g and A((95,97)Mo) matrices nearly identical to those of SO, indicating a similar pentacoordinate active site in SoxCD1. However, no sulfite-induced reduction to Mo(V) was detected, nor could a strongly coupled (1)H signal or a phosphate-inhibited species be generated. This indicates that the outer coordination sphere controls substrate binding in SoxCD, permitting access only to protein-bound sulfur via the C-terminal tail of SoxY.

Leimkuhler S, Wuebbens MM, Rajagopalan KV
The History of the Discovery of the Molybdenum Cofactor and Novel Aspects of its Biosynthesis in Bacteria.
Coord Chem Rev. 2011; 255: 1129-1144
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Biosynthesis of the molybdenum cofactor in bacteria is described with a detailed analysis of each individual reaction leading to the formation of stable intermediates during the synthesis of molybdopterin from GTP. As a starting point, the discovery of molybdopterin and the elucidation of its structure through the study of stable degradation products are described. Subsequent to molybdopterin synthesis, the molybdenum atom is added to the molybdopterin dithiolene group to form the molybdenum cofactor. This cofactor is either inserted directly into specific molybdoenzymes or is further modified by the addition of nucleotides to the molybdopterin phosphate group or the replacement of ligands at the molybdenum center.

Wilcoxen J, Snider S, Hille R
Substitution of silver for copper in the binuclear Mo/Cu center of carbon monoxide dehydrogenase from Oligotropha carboxidovorans.
J Am Chem Soc. 2011; 133: 12934-6
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Carbon monoxide dehydrogenase from Oligotropha carboxidovorans catalyzes the aerobic oxidation of carbon monoxide to carbon dioxide, providing the organism both a carbon source and energy for growth. The active site of the native enzyme is a unique binuclear molybdenum- and copper-containing center. Here we show that silver can be substituted for copper in the active site to yield a functional enzyme. The characteristic hyperfine coupling of the I = (1/2) nucleus of Ag is evident in the EPR signal of the binuclear active site observed upon reduction with CO, indicating both the incorporation of silver into the active site and, remarkably, retention of the catalytic activity. The silver-substituted enzyme is reduced by CO with an observed limiting rate constant of 8.1 s(-1), which can be compared with the value of 51 s(-1) for the wild-type enzyme. Steady-state kinetics for the Ag-substituted enzyme yielded k(cat) = 8.2 s(-1) and K(m) = 2.95 muM at pH 7.2.

Hille R, Nishino T, Bittner F
Molybdenum enzymes in higher organisms.
Coord Chem Rev. 2011; 255: 1179-1205
Display abstract
Recent progress in our understanding of the structural and catalytic properties of molybdenum-containing enzymes in eukaryotes is reviewed, along with aspects of the biosynthesis of the cofactor and its insertion into apoprotein.

Spreitler F, Brock C, Pelzmann A, Meyer O, Kohler J
Interaction of CO dehydrogenase with the cytoplasmic membrane monitored by fluorescence correlation spectroscopy.
Chembiochem. 2010; 11: 2419-23
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We have investigated the interaction of carbon monoxide dehydrogenase (CODH), an enzyme that catalyses the oxidation of CO in the aerobic eubacterium Oligotropha carboxidovorans, with the cytoplasmic membrane by using fluorescence correlation spectroscopy (FCS). Our results reveal that in vitro this interaction of CODH is specific for cytoplasmic membranes from CO-grown bacteria.

Ryde U, Schulzke C, Starke K
Which functional groups of the molybdopterin ligand should be considered when modeling the active sites of the molybdenum and tungsten cofactors? A density functional theory study.
J Biol Inorg Chem. 2009; 14: 1053-64
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A density functional theory study of the influence of the various functional groups of the molybdopterin ligand on electronic and geometric properties of active-site models for the molybdenum and tungsten cofactors has been undertaken. We used analogous molybdenum and tungsten complexes with increasingly accurate representation of the molybdopterin ligands and compared bond lengths, angles, charge distribution, composition of the binding orbitals, as well as the redox potentials in relation to each other. On the basis of our findings, we suggest using ligand systems including the pyrane and the pyrazine rings, besides the dithiolene function, to obtain sufficiently reliable computational, but also synthetic, models for the molybdenum and tungsten cofactors, whereas the second ring of the pterin might be neglected for efficiency reasons.

Groysman S, Holm RH
Biomimetic chemistry of iron, nickel, molybdenum, and tungsten in sulfur-ligated protein sites.
Biochemistry. 2009; 48: 2310-20
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Biomimetic inorganic chemistry has as its primary goal the synthesis of molecules that approach or achieve the structures, oxidation states, and electronic and reactivity features of native metal-containing sites of variant nuclearity. Comparison of properties of accurate analogues and these sites ideally provides insight into the influence of protein structure and environment on intrinsic properties as represented by the analogue. For polynuclear sites in particular, the goal provides a formidable challenge for, with the exception of iron-sulfur clusters, all such site structures have never been achieved and few have even been closely approximated by chemical synthesis. This account describes the current status of the synthetic analogue approach as applied to the mononuclear sites in certain molybdoenzymes and the polynuclear sites in hydrogenases, nitrogenase, and carbon monoxide dehydrogenases.

Hernandez JA, George SJ, Rubio LM
Molybdenum trafficking for nitrogen fixation.
Biochemistry. 2009; 48: 9711-21
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The molybdenum nitrogenase is responsible for most biological nitrogen fixation, a prokaryotic metabolic process that determines the global biogeochemical cycles of nitrogen and carbon. Here we describe the trafficking of molybdenum for nitrogen fixation in the model diazotrophic bacterium Azotobacter vinelandii. The genes and proteins involved in molybdenum uptake, homeostasis, storage, regulation, and nitrogenase cofactor biosynthesis are reviewed. Molybdenum biochemistry in A. vinelandii reveals unexpected mechanisms and a new role for iron-sulfur clusters in the sequestration and delivery of molybdenum.

Yabe T et al.
Structural analysis of Arabidopsis CnfU protein: an iron-sulfur cluster biosynthetic scaffold in chloroplasts.
J Mol Biol. 2008; 381: 160-73
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CnfU, a key iron-sulfur (Fe-S) cluster biosynthetic scaffold that is required for biogenesis of ferredoxin and photosystem I in chloroplasts, consists of two tandemly repeated domains in which only the N-terminal domain contains a conserved CXXC motif. We have determined the crystal structure of the metal-free dimer of AtCnfU-V from Arabidopsis thaliana at 1.35 A resolution. The N-terminal domains of the two monomers are linked together through two intermolecular disulfide bonds between the CXXC motifs. At the dimer interface, a total of four cysteine sulfur atoms provide a Fe-S cluster assembly site surrounded by uncharged but hydrophilic structurally mobile segments. The C-terminal domain of one monomer interacts with the N-terminal domain of the opposing monomer and thereby stabilizes dimer formation. Furthermore, Fe K-edge X-ray absorption spectroscopic analysis of the holo-CnfU dimer in solution suggests the presence of a typical [2Fe-2S]-type cluster coordinated by four thiolate ligands. Based on these data, a plausible model of the holo-AtCnfU-V dimer containing a surface-exposed [2Fe-2S] cluster assembled in the dimer interface was deduced. We propose that such a structural framework is important for CnfU to function as a Fe-S cluster biosynthetic scaffold.

Neumann M, Leimkuhler S
Heavy metal ions inhibit molybdoenzyme activity by binding to the dithiolene moiety of molybdopterin in Escherichia coli.
FEBS J. 2008; 275: 5678-89
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Molybdenum insertion into the dithiolene group on the 6-alkyl side-chain of molybdopterin is a highly specific process that is catalysed by the MoeA and MogA proteins in Escherichia coli. Ligation of molybdate to molybdopterin generates the molybdenum cofactor, which can be inserted directly into molybdoenzymes binding the molybdopterin form of the molybdenum cofactor, or is further modified in bacteria to form the dinucleotide form of the molybdenum cofactor. The ability of various metals to bind tightly to sulfur-rich sites raised the question of whether other metal ions could be inserted in place of molybdenum at the dithiolene moiety of molybdopterin in molybdoenzymes. We used the heterologous expression systems of human sulfite oxidase and Rhodobacter sphaeroides dimethylsulfoxide reductase in E. coli to study the incorporation of different metal ions into the molybdopterin site of these enzymes. From the added metal-containing compounds Na(2)MoO(4), Na(2)WO(4), NaVO(3), Cu(NO(3))(2), CdSO(4) and NaAsO(2) during the growth of E. coli, only molybdate and tungstate were specifically inserted into sulfite oxidase and dimethylsulfoxide reductase. Other metals, such as copper, cadmium and arsenite, were nonspecifically inserted into sulfite oxidase, but not into dimethylsulfoxide reductase. We showed that metal insertion into molybdopterin occurs beyond the step of molybdopterin synthase and is independent of MoeA and MogA proteins. Our study shows that the activity of molybdoenzymes, such as sulfite oxidase, is inhibited by high concentrations of heavy metals in the cell, which will help to further the understanding of metal toxicity in E. coli.

Aragao D, Mitchell EP, Frazao CF, Carrondo MA, Lindley PF
Structural and functional relationships in the hybrid cluster protein family: structure of the anaerobically purified hybrid cluster protein from Desulfovibrio vulgaris at 1.35 A resolution.
Acta Crystallogr D Biol Crystallogr. 2008; 64: 665-74
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The hybrid cluster protein (HCP) from the sulfate-reducing bacterium Desulfovibrio vulgaris strain Hildenborough has been isolated and crystallized anaerobically. The phase problem was solved for a P2(1)2(1)2(1) crystal form using multiple-wavelength anomalous diffraction data collected in the vicinity of the Fe K absorption edge. Although the overall protein structure is essentially the same as that previously obtained, it shows that the nature of the hybrid cluster has particular differences when isolated and crystallized in the absence of oxygen and this provides insight into the structural features associated with changes in the oxidation state. A comparison between HCPs and carbon monoxide dehydrogenases (CoDs) shows that they possess a similar fold and that the dehydrogenases have a related cluster at the equivalent HCP hybrid cluster position. This helps to understand the nature of the hybrid cluster and to predict a dimeric structure for class 3 HCPs, which lack the N-terminal region.

Johannes J, Unciuleac MC, Friedrich T, Warkentin E, Ermler U, Boll M
Inhibitors of the molybdenum cofactor containing 4-hydroxybenzoyl-CoA reductase.
Biochemistry. 2008; 47: 4964-72
Display abstract
4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a member of the xanthine oxidase (XO) family of molybdenum cofactor containing enzymes and catalyzes the irreversible removal of a phenolic hydroxy group by reduction, yielding benzoyl-CoA and water. In this work the effects of various activity modulating compounds were characterized by kinetic, electron paramagnetic resonance (EPR) spectroscopic, and X-ray crystallographic studies. 4-HBCR was readily inactivated by cyanide and by the reducing agents titanium(III) citrate and dithionite; in contrast, reduced viologens had no inhibitory effect. Cyanide inhibition occurred in both the oxidized and reduced state of 4-HBCR. In the reduced state, cyanide-inhibited 4-HBCR was reactivated by simple oxidation. In contrast, reactivation from the oxidized state was only achieved in the presence of sulfide. Dithionite-inhibited 4-HBCR was reactivated by oxidation, whereas inhibition by titanium(III) citrate was irreversible. The previously reported inhibitory effect of azide could not be confirmed; instead, azide rather protected the enzyme from inactivation by titanium(III) citrate. The EPR spectra of the Mo(V) states were nearly identical in the noninhibited methyl viologen and in the dithionite-inhibited states of 4-HBCR; they exhibited a hyperfine splitting due to magnetic coupling with two solvent-exchangeable protons. The cyanide-treated enzyme showed the typical desulfo-inhibited Mo(V) EPR signal in D 2O, whereas in H 2O the hyperfine splitting was altered but indicated no loss of Mo(V)-proton interactions. The structures of dithionite- and azide-bound 4-HBCR were solved at 2.1 and 2.2 A, respectively. Both dithionite and azide bound directly to equatorial ligation sites of the Mo atom. The results obtained revealed further insights into the active site of an unusual member of the XO family of molybdenum cofactor containing enzymes.

Mendel RR
Biology of the molybdenum cofactor.
J Exp Bot. 2007; 58: 2289-96
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The transition element molybdenum (Mo) is an essential micronutrient for plants where it is needed as a catalytically active metal during enzyme catalysis. Four plant enzymes depend on molybdenum: nitrate reductase, sulphite oxidase, xanthine dehydrogenase, and aldehyde oxidase. However, in order to gain biological activity and fulfil its function in enzymes, molybdenum has to be complexed by a pterin compound thus forming the molybdenum cofactor. In this article, the path of molybdenum from its uptake into the cell, via formation of the molybdenum cofactor and its storage, to the final modification of the molybdenum cofactor and its insertion into apo-metalloenzymes will be reviewed.

Burgmayer SJ et al.
Synthesis, characterization, and spectroscopy of model molybdopterin complexes.
J Inorg Biochem. 2007; 101: 1601-16
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The preparation and characterization of new model complexes for the molybdenum cofactor are reported. The new models are distinctive for the inclusion of pterin-substituted dithiolene chelates and have the formulation Tp(*)MoX(pterin-R-dithiolene) (Tp(*)=tris(3,5,-dimethylpyrazolyl)borate), X=O, S, R=aryl. Syntheses of Mo(4+) and (5+) complexes of two pterin-dithiolene derivatives as both oxo and sulfido compounds, and improved syntheses for pterinyl alkynes and [Et(4)N][Tp(*)Mo(IV)(S)S(4)] reagents are described. Characterization methods include electrospray ionization mass spectrometry, electrochemistry, infrared spectroscopy, electron paramagnetic resonance and magnetic circular dichroism. Cyclic voltammetry reveals that the Mo(5+/4+) reduction potential is intermediate between that for dithiolenes with electron-withdrawing substituents and simple dithiolates chelates. Electron paramagnetic resonance and magnetic circular dichroism of Mo(5+) complexes where X=O, R=aryl indicates that the molybdenum environment in the new models is electronically similar to that in Tp(*)MoO(benzenedithiolate).

Jeoung JH, Dobbek H
Carbon dioxide activation at the Ni,Fe-cluster of anaerobic carbon monoxide dehydrogenase.
Science. 2007; 318: 1461-4
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Anaerobic CO dehydrogenases catalyze the reversible oxidation of CO to CO2 at a complex Ni-, Fe-, and S-containing metal center called cluster C. We report crystal structures of CO dehydrogenase II from Carboxydothermus hydrogenoformans in three different states. In a reduced state, exogenous CO2 supplied in solution is bound and reductively activated by cluster C. In the intermediate structure, CO2 acts as a bridging ligand between Ni and the asymmetrically coordinated Fe, where it completes the square-planar coordination of the Ni ion. It replaces a water/hydroxo ligand bound to the Fe ion in the other two states. The structures define the mechanism of CO oxidation and CO2 reduction at the Ni-Fe site of cluster C.

Pacher P, Nivorozhkin A, Szabo C
Therapeutic effects of xanthine oxidase inhibitors: renaissance half a century after the discovery of allopurinol.
Pharmacol Rev. 2006; 58: 87-114
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The prototypical xanthine oxidase (XO) inhibitor allopurinol, has been the cornerstone of the clinical management of gout and conditions associated with hyperuricemia for several decades. More recent data indicate that XO also plays an important role in various forms of ischemic and other types of tissue and vascular injuries, inflammatory diseases, and chronic heart failure. Allopurinol and its active metabolite oxypurinol showed considerable promise in the treatment of these conditions both in experimental animals and in small-scale human clinical trials. Although some of the beneficial effects of these compounds may be unrelated to the inhibition of the XO, the encouraging findings rekindled significant interest in the development of additional, novel series of XO inhibitors for various therapeutic indications. Here we present a critical overview of the effects of XO inhibitors in various pathophysiological conditions and also review the various emerging therapeutic strategies offered by this approach.

Fischer K et al.
Function and structure of the molybdenum cofactor carrier protein from Chlamydomonas reinhardtii.
J Biol Chem. 2006; 281: 30186-94
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The molybdenum cofactor (Moco) forms the catalytic site in all eukaryotic molybdenum enzymes and is synthesized by a multistep biosynthetic pathway. The mechanism of transfer, storage, and insertion of Moco into the appropriate apo-enzyme is poorly understood. In Chlamydomonas reinhardtii, a Moco carrier protein (MCP) has been identified and characterized recently. Here we show biochemical evidence that MCP binds Moco as well as the tungstate-substituted form of the cofactor (Wco) with high affinity, whereas molybdopterin, the ultimate cofactor precursor, is not bound. This binding selectivity points to a specific metal-mediated interaction with MCP, which protects Moco and Wco from oxidation with t((1/2)) of 24 and 96 h, respectively. UV-visible spectroscopy showed defined absorption bands at 393, 470, and 570 nm pointing to ene-diothiolate and protein side-chain charge transfer bonds with molybdenum. We have determined the crystal structure of MCP at 1.6 Angstrom resolution using seleno-methionated and native protein. The monomer constitutes a Rossmann fold with two homodimers forming a symmetrical tetramer in solution. Based on conserved surface residues, charge distribution, shape, in silico docking studies, structural comparisons, and identification of an anionbinding site, a prominent surface depression was proposed as a Moco-binding site, which was confirmed by structure-guided mutagenesis coupled to substrate binding studies.

Resch M, Dobbek H, Meyer O
Structural and functional reconstruction in situ of the [CuSMoO2] active site of carbon monoxide dehydrogenase from the carbon monoxide oxidizing eubacterium Oligotropha carboxidovorans.
J Biol Inorg Chem. 2005; 10: 518-28
Display abstract
Carbon monoxide dehydrogenase from the bacterium Oligotropha carboxidovorans catalyzes the oxidation of CO to CO(2) at a unique [CuSMoO(2)] cluster. In the bacteria the cluster is assembled post-translational. The integration of S, and particularly of Cu, is rate limiting in vivo, which leads to CO dehydrogenase preparations containing the mature and fully functional enzyme along with forms of the enzyme deficient in one or both of these elements. The active sites of mature and immature forms of CO dehydrogenase were converted into a [MoO(3)] centre by treatment with potassium cyanide. We have established a method, which rescues 50% of the CO dehydrogenase activity by in vitro reconstitution of the active site through the supply of sulphide first and subsequently of Cu(I) under reducing conditions. Immature forms of CO dehydrogenase isolated from the bacterium, which were deficient in S and/or Cu at the active site, were similarly activated. X-ray crystallography and electron paramagnetic resonance spectroscopy indicated that the [CuSMoO(2)] cluster was properly reconstructed. However, reconstituted CO dehydrogenase contains mature along with immature forms. The chemical reactions of the reconstitution of CO dehydrogenase are summarized in a model, which assumes resulphuration of the Mo-ion at both equatorial positions at a 1:1 molar ratio. One equatorial Mo-S group reacts with Cu(I) in a productive fashion yielding a mature, functional [CuSMoO(2)] cluster. The other Mo-S group reacts with Cu(I), then Cu(2)S is released and an oxo group is introduced from water, yielding an inactive [MoO(3)] centre.

Heidenreich T, Wollers S, Mendel RR, Bittner F
Characterization of the NifS-like domain of ABA3 from Arabidopsis thaliana provides insight into the mechanism of molybdenum cofactor sulfuration.
J Biol Chem. 2005; 280: 4213-8
Display abstract
The molybdenum cofactor sulfurase ABA3 from Arabidopsis thaliana specifically regulates the activity of the molybdenum enzymes aldehyde oxidase and xanthine dehydrogenase by converting their molybdenum cofactor from the desulfo-form into the sulfo-form. ABA3 is a two-domain protein with an NH2-terminal domain sharing significant similarities to NifS proteins that catalyze the decomposition of l-cysteine to l-alanine and elemental sulfur for iron-sulfur cluster synthesis. Although different in its physiological function, the mechanism of ABA3 for sulfur mobilization was found to be similar to NifS proteins. The protein binds a pyridoxal phosphate cofactor and a substrate-derived persulfide intermediate, and site-directed mutagenesis of strictly conserved binding sites for the cofactor and the persulfide demonstrated that they are essential for molybdenum cofactor sulfurase activity. In vitro, the NifS-like domain of ABA3 activates aldehyde oxidase and xanthine dehydrogenase in the absence of the C-terminal domain, but in vivo, the C-terminal domain is required for proper activation of both target enzymes. In addition to its cysteine desulfurase activity, ABA3-NifS also exhibits selenocysteine lyase activity. Although l-selenocysteine is unlikely to be a natural substrate for ABA3, it is decomposed more efficiently than l-cysteine. Besides mitochondrial AtNFS1 and plastidial AtNFS2, which are both proposed to be involved in iron-sulfur cluster formation, ABA3 is proposed to be a third and cytosolic NifS-like cysteine desulfurase in A. thaliana. However, the sulfur transferase activity of ABA3 is used for post-translational activation of molybdenum enzymes rather than for iron-sulfur cluster assembly.

Takuma M, Ohki Y, Tatsumi K
Sulfido-bridged dinuclear molybdenum-copper complexes related to the active site of CO dehydrogenase: [(dithiolate)Mo(O)S2Cu(SAr)]2- (dithiolate = 1,2-S2C6H4, 1,2-S2C6H2-3,6-Cl2, 1,2-S2C2H4).
Inorg Chem. 2005; 44: 6034-43
Display abstract
The [MoCu] carbon monoxide dehydrogenase (CODH) is a Cu-containing molybdo-flavoprotein, the active site of which contains a pterin-dithiolene cofactor bound to a sulfido-bridged dinuclear Mo-Cu complex. In this paper, the synthesis and characterization of dinuclear Mo-Cu complexes relevant to the active site of [MoCu]-CODH are described. Reaction of [MoO2S2]2- with CuCN affords the dinuclear complex [O2MoS2Cu(CN)]2- (1), in which the CN- ligand can be replaced with various aryl thiolates to give rise to a series of dinuclear complexes [O2MoS2Cu(SAr)]2- (Ar = Ph (2), o-Tol (3), and p-Tol (4)). An alternative synthesis of complex 2 is the reaction of [MoO2S2]2- with [Cu(SPh)3]2-. Similarly, [O2MoS2Cu(PPh3)]- (5), [O2MoS2Cu(dppe)]- (dppe = 1,2-bis(diphenylphosphino)ethane) (6), and [O2MoS2Cu(triphos)]- (triphos = 1,1,1-tris[(diphenylphosphino)methyl]ethane) (7) were prepared from the reactions of [MoO2S2]2- with the Cu(I) phosphine complexes. Treatment of 1, 2, 4, or 5 with dithiols (1,2-(SH)2C6H4, 1,2-(SH)2C6H2-3,6-Cl2, and 1,2-(SH)2C2H4), in acetonitrile, leads to the replacement of a molybdenum-bound oxo ligand to yield [(dithiolate)Mo(O)S2CuL]2- (L = CN, SAr; dithiolate = 1,2-S2C6H4, 1,2-S2C6H2-3,6-Cl2, or 1,2-S2C2H4) (8-13) or [(1,2-S2C6H4)Mo(O)S2Cu(PPh3)]- (14) complexes.

Boer DR, Muller A, Fetzner S, Lowe DJ, Romao MJ
On the purification and preliminary crystallographic analysis of isoquinoline 1-oxidoreductase from Brevundimonas diminuta 7.
Acta Crystallogr Sect F Struct Biol Cryst Commun. 2005; 61: 137-40
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Isoquinoline 1-oxidoreductase (IOR) from Brevundimonas diminuta is a mononuclear molybdoenzyme of the xanthine-dehydrogenase family of proteins and catalyzes the conversion of isoquinoline to isoquinoline-1-one. Its primary sequence and behaviour, specifically in its substrate specificity and lipophilicity, differ from other members of the family. A crystal structure of the enzyme is expected to provide an explanation for these differences. This paper describes the crystallization and preliminary X-ray diffraction experiments as well as an optimized purification protocol for IOR. Crystallization of IOR was achieved using two different crystallization buffers. Streak-seeding and cross-linking were essential to obtain well diffracting crystals. Suitable cryo-conditions were found and a structure solution was obtained by molecular replacement. However, phases need to be improved in order to obtain a more interpretable electron-density map.

Agnelli P et al.
The unexpected structural role of glutamate synthase [4Fe-4S](+1,+2) clusters as demonstrated by site-directed mutagenesis of conserved C residues at the N-terminus of the enzyme beta subunit.
Arch Biochem Biophys. 2005; 436: 355-66
Display abstract
Azospirillum brasilense glutamate synthase (GltS) is a complex iron-sulfur flavoprotein whose catalytically active alphabeta protomer (alpha subunit, 162kDa; beta subunit, 52.3 kDa) contains one FAD, one FMN, one [3Fe-4S](0,+1), and two [4Fe-4S](+1,+2) clusters. The structure of the alpha subunit has been determined providing information on the mechanism of ammonia transfer from L-glutamine to 2-oxoglutarate through a 30 A-long intramolecular tunnel. On the contrary, details of the electron transfer pathway from NADPH to the postulated 2-iminoglutarate intermediate through the enzyme flavin co-factors and [Fe-S] clusters are largely indirect. To identify the location and role of each one of the GltS [4Fe-4S] clusters, we individually substituted the four cysteinyl residues forming the first of two conserved C-rich regions at the N-terminus of GltS beta subunit with alanyl residues. The engineered genes encoding the beta subunit variants (and derivatives carrying C-terminal His6-tags) were co-expressed with the wild-type alpha subunit gene. In all cases the C/A substitutions prevented alpha and beta subunits association to yield the GltS alphabeta protomer. This result is consistent with the fact that these residues are responsible for the formation of glutamate synthase [4Fe-4S](+1,+2) clusters within the N-terminal region of the beta subunit, and that these clusters are implicated not only in electron transfer between the GltS flavins, but also in alphabeta heterodimer formation by structuring an N-terminal [Fe-S] beta subunit interface subdomain, as suggested by the three-dimensional structure of dihydropyrimidine dehydrogenase, an enzyme containing an N-terminal beta subunit-like domain.

Hemann C, Ilich P, Stockert AL, Choi EY, Hille R
Resonance Raman studies of xanthine oxidase: The reduced enzyme-product complex with violapterin.
J Phys Chem B. 2005; 109: 3023-31
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A study of the molecular, electronic, and vibrational characteristics of the molybdenum-containing enzyme complex xanthine oxidase with violapterin has been carried out using density functional theory calculations and resonance Raman spectroscopy. The electronic structure calculations were carried out on a model consisting of the enzyme molybdopterin cofactor [in the four-valent, reduced state; Mo(IV)O(SH)] covalently linked to violapterin (1H,3H,8H-pteridine-2,4,7-trione in the neutral form) via an oxygen bridge, Mo-O-C7. Full geometry optimizations were performed for all models using the SDD basis set and the three-parameter exchange functional of Becke combined with the Lee, Yang, and Parr correlational functional. Harmonic vibrational frequencies were determined for a variety of isotopes in an attempt to correlate experimentally observed shifts upon 18O-labeling of the Mo-OR bridge to bound product as well as shifts seen upon substitution of solvent-exchangeable protons in samples prepared in D2O. The theoretical vibrational frequencies compared favorably with experimentally observed vibrational modes in the resonance Raman spectra of the reduced xanthine oxidase-violapterin complex prepared in H2O and D2O and with 18O-labeled product. Correlating the isotopic shifts from the calculations with those from the resonance Raman experiments resulted in complete normal mode assignments for all modes observed in the 350-1750 cm(-1) range. The present work demonstrates that a model in which the violapterin is coordinated to the molybdenum of the active site in a simple end-on manner via the hydroxyl group introduced by an enzyme accurately predicts the observed resonance Raman spectrum of the complex. Given the numerous modes involving the bridging oxygen, a side-on binding mode can be eliminated.

Panda R et al.
Initial structure modification of tetrahedral to planar nickel(II) in a nickel-iron-sulfur cluster related to the C-cluster of carbon monoxide dehydrogenase.
J Am Chem Soc. 2004; 126: 6448-59
Display abstract
A method has been devised that creates a planar Ni(II) site from a tetrahedral site in a NiFe(3)S(4) cubane-type cluster. Reaction of [(Ph(3)P)NiFe(3)S(4)(LS(3))](2)(-) (2) with 1,2-bis(dimethylphosphino)ethane affords [(dmpe)NiFe(3)S(4)(LS(3))](2)(-) (3), isolated in ca. 45% yield as (Et(4)N)(2)[3a].2.5MeCN and (Et(4)N)(2)[3b].0.25MeCN, both of which occur in triclinic space group P. Each crystalline form contains two crystallographically inequivalent clusters with the same overall structure but slightly different dimensions. The cluster is bound by three thiolate terminal ligands to semirigid cavitand ligand LS(3). The NiFe(3)S(4) core contains three tetrahedral sites, one Fe(micro(3)-S)(3)(SR) and two Fe(micro(3)-S)(2)(micro(2)-S)(SR) with normal metric features, and one distorted square planar Ni(micro(3)-S)(2)P(2) site in a Ni(micro(3)-S)(2)Fe face with mean bond lengths Ni-P = 2.147(9) A and Ni-S = 2.29(2) A. The opposite Fe(2)(micro(3)-S)(micro(2)-S) face places the micro(2)-S atom at nonbonding and variable distances (2.60-2.90 A) above the nickel atom. Binding of the strong-field ligand dmpe results in a planar Ni(II) site and deconstruction of the full cubane geometry. The structure approximates that established crystallographically in the C-cluster of C. hydrogenoformans carbon monoxide dehydrogenase whose NiFe(4)S(4) core contains a planar NiS(4) site and three tetrahedral FeS(4) sites in a fragment that is bridged by sulfide atoms to an exo iron atom. Mossbauer studies of polycrystalline samples containing both clusters 3a and 3b reveal the presence of at least two cluster types. The spectroscopically best defined cluster accounts for ca. 54% of total iron and exhibits hyperfine interactions quite similar to those reported for the S = (5)/(2) state of the protein-bound cubane-type cluster [ZnFe(3)S(4)](1+), whose Mossbauer spectrum revealed the presence of a high-spin Fe(2+) site and a delocalized Fe(2.5+)Fe(2.5+) pair. Development of reactions leading to a planar nickel and a sulfide-bridged iron atom is requisite to attainment of a synthetic analogue of this complex protein-bound cluster. This work demonstrates a tetrahedral (2) --> planar (3) Ni(II) stereochemical conversion can be effected by binding of ligands that generate a sufficiently strong in-plane ligand field (dmpe = 1,2-bis(dimethylphosphino)ethane, LS(3) = 1,3,5-tris((4,6-dimethyl-3-mercaptophenyl)thio)-2,4,6-tris(p-tolylthio)benzene(3- )).

Roberts GP, Youn H, Kerby RL
CO-sensing mechanisms.
Microbiol Mol Biol Rev. 2004; 68: 453-73
Display abstract
Carbon monoxide (CO) has long been known to have dramatic physiological effects on organisms ranging from bacteria to humans, but recently there have a number of suggestions that organisms might have specific sensors for CO. This article reviews the current evidence for a variety of proteins with demonstrated or potential CO-sensing ability. Particular emphasis is placed on the molecular description of CooA, a heme-containing CO sensor from Rhodospirillum rubrum, since its biological role as a CO sensor is clear and we have substantial insight into the basis of its sensing ability.

Bader G, Gomez-Ortiz M, Haussmann C, Bacher A, Huber R, Fischer M
Structure of the molybdenum-cofactor biosynthesis protein MoaB of Escherichia coli.
Acta Crystallogr D Biol Crystallogr. 2004; 60: 1068-75
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The moaABC operon of Escherichia coli is involved in early steps of the biosynthesis of the molybdenum-binding cofactor molybdopterin, but the precise functions of the cognate proteins are not known. The crystal structure of the MoaB protein from E. coli was determined by multiple anomalous dispersion at 2.1 angstroms A resolution and refined to an R factor of 20.4% (Rfree = 25.0%). The protein is a 32-symmetric hexamer, with the monomers consisting of a central beta-sheet flanked by helices on both sides. The overall fold of the monomer is similar to those of the MogA protein of E. coli, the G-domains of rat and human gephyrin and the G-domains of Cnx1 protein from A. thaliana, all of which are involved in the insertion of an unknown molybdenum species into molybdopterin to form the molybdenum cofactor. Furthermore, the MoaB protein shows significant sequence similarity to the cinnamon protein from Drosophila melanogaster. In addition to other functions, all these proteins are involved in the biosynthesis of the molybdenum cofactor and have been shown to bind molybdopterin. The close structural homology to MogA and the gephyrin and Cnx1 domains suggests that MoaB may bind a hitherto unidentified pterin compound, possibly an intermediate in molybdopterin biosynthesis.

Correia dos Santos MM, Sousa PM, Goncalves ML, Romao MJ, Moura I, Moura JJ
Direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase.
Eur J Biochem. 2004; 271: 1329-38
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This work reports on the direct electrochemistry of the Desulfovibrio gigas aldehyde oxidoreductase (DgAOR), a molybdenum enzyme of the xanthine oxidase family that contains three redox-active cofactors: two [2Fe-2S] centers and a molybdopterin cytosine dinucleotide cofactor. The voltammetric behavior of the enzyme was analyzed at gold and carbon (pyrolytic graphite and glassy carbon) electrodes. Two different strategies were used: one with the molecules confined to the electrode surface and a second with DgAOR in solution. In all of the cases studied, electron transfer took place, although different redox reactions were responsible for the voltammetric signal. From a thorough analysis of the voltammetric responses and the structural properties of the molecular surface of DgAOR, the redox reaction at the carbon electrodes could be assigned to the reduction of the more exposed iron cluster, [2Fe-2S] II, whereas reduction of the molybdopterin cofactor occurs at the gold electrode. Voltammetric results in the presence of aldehydes are also reported and discussed.

Berry CE, Hare JM
Xanthine oxidoreductase and cardiovascular disease: molecular mechanisms and pathophysiological implications.
J Physiol. 2004; 555: 589-606
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There is substantial evidence that oxidative stress participates in the pathophysiology of cardiovascular disease. Biochemical, molecular and pharmacological studies further implicate xanthine oxidoreductase (XOR) as a source of reactive oxygen species in the cardiovascular system. XOR is a member of the molybdoenzyme family and is best known for its catalytic role in purine degradation, metabolizing hypoxanthine and xanthine to uric acid with concomitant generation of superoxide. Gene expression of XOR is regulated by oxygen tension, cytokines and glucocorticoids. XOR requires molybdopterin, iron-sulphur centres, and FAD as cofactors and has two interconvertible forms, xanthine oxidase and xanthine dehydrogenase, which transfer electrons from xanthine to oxygen and NAD(+), respectively, yielding superoxide, hydrogen peroxide and NADH. Additionally, XOR can generate superoxide via NADH oxidase activity and can produce nitric oxide via nitrate and nitrite reductase activities. While a role for XOR beyond purine metabolism was first suggested in ischaemia-reperfusion injury, there is growing awareness that it also participates in endothelial dysfunction, hypertension and heart failure. Importantly, the XOR inhibitors allopurinol and oxypurinol attenuate dysfunction caused by XOR in these disease states. Attention to the broader range of XOR bioactivity in the cardiovascular system has prompted initiation of several randomised clinical outcome trials, particularly for congestive heart failure. Here we review XOR gene structure and regulation, protein structure, enzymology, tissue distribution and pathophysiological role in cardiovascular disease with an emphasis on heart failure.

Rees DC
Great metalloclusters in enzymology.
Annu Rev Biochem. 2002; 71: 221-46
Display abstract
Metallocluster-containing enzymes catalyze some of the most basic redox transformations in the biosphere. The reactions catalyzed by these enzymes typically involve small molecules such as N2, CO, and H2 that are used to generate both chemical building blocks and energy for metabolic purposes. During the past decade, structures have been established for the iron-sulfur-based metalloclusters present in the molybdenum nitrogenase, the iron-only hydrogenase, and the nickel-carbon monoxide dehydrogenase, and for the copper-sulfide-based cluster in nitrous oxide reductase. Although these clusters are built from interactions observed in simpler metalloproteins, they contain novel features that may be relevant for their catalytic function. The mechanisms of metallocluster-containing enzymes are still poorly defined, and represent substantial and continuing challenges to biochemists, biophysicists, and synthetic chemists. These proteins also provide a window into the union of the biological and inorganic worlds that may have been relevant to the early evolution of biochemical catalysis.

Muller J, Lugovskoy AA, Wagner G, Lippard SJ
NMR structure of the [2Fe-2S] ferredoxin domain from soluble methane monooxygenase reductase and interaction with its hydroxylase.
Biochemistry. 2002; 41: 42-51
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The soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) is a multicomponent enzyme system required for the conversion of methane to methanol. It comprises a hydroxylase, a regulatory protein, and a reductase. The reductase contains two domains: an NADH-binding and FAD-containing flavin domain and a ferredoxin (Fd) domain carrying a [2Fe-2S] cofactor. Here, we report the solution structure of the reduced form of the 98-amino acid Fd domain (Blazyk, J. L., and Lippard, S. J. Unpublished results) determined by nuclear magnetic resonance (NMR) spectroscopy and restrained molecular dynamics calculations. The structure consists of six beta strands arranged into two beta sheets as well as three alpha helices. Two of these helices form a helix-proline-helix motif, unprecedented among [2Fe-2S] proteins. The [2Fe-2S] cluster is coordinated by the sulfur atoms of cysteine residues 42, 47, 50, and 82. The 10.9 kDa ferredoxin domain of the reductase protein transfers electrons to carboxylate-bridged diiron centers in the 251 kDa hydroxylase component of sMMO. The binding of the Fd domain with the hydroxylase was investigated by NMR spectroscopy. The hydroxylase binding surface on the ferredoxin protein has a polar center surrounded by patches of hydrophobic residues. This arrangement of amino acids differs from that by which previously studied [2Fe-2S] proteins interact with their electron-transfer partners. The critical residues on the Fd domain involved in this binding interaction map well onto the universally conserved residues of sMMO enzymes from different species. We propose that the [2Fe-2S] domains in these other sMMO systems have a fold very similar to the one found here for M. capsulatus (Bath) MMOR-Fd.

Delarbre L, Stevenson CE, White DJ, Mitchenall LA, Pau RN, Lawson DM
Two crystal structures of the cytoplasmic molybdate-binding protein ModG suggest a novel cooperative binding mechanism and provide insights into ligand-binding specificity.
J Mol Biol. 2001; 308: 1063-79
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The X-ray structures of the cytoplasmic molybdate-binding protein ModG from Azotobacter vinelandii in two different crystal forms have been determined. For such a small protein it is remarkably complex. Each 14.3 kDa subunit contains two small beta-barrel domains, which display an OB-fold motif, also seen in the related structure of ModE, a molybdenum-dependent transcriptional regulator, and very recently in the Mop protein that, like ModG, has been implicated in molybdenum homeostasis within the cell. In contrast to earlier speculation, the functional unit of ModG is actually not a dimer (as in ModE), but a trimer capable of binding a total of eight molybdate molecules that are distributed between two disparate types of site. All the binding sites are located at subunit interfaces, with one type lying on a crystallographic 3-fold axis, whilst the other lies between pairs of subunits. The two types of site are linked by short hydrogen bond networks that may suggest a cooperative binding mechanism. A superposition of two subunits of the ModG trimer on the apo-ModE dimer allows the probable locations of the molybdate-binding sites of the latter to be assigned. Through structural comparisons with other oxyanion-binding proteins, including Mop and ModE, it is possible to speculate about ligand-binding affinities, selectivity and evolution.

Boll M et al.
Redox centers of 4-hydroxybenzoyl-CoA reductase, a member of the xanthine oxidase family of molybdenum-containing enzymes.
J Biol Chem. 2001; 276: 47853-62
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4-Hydroxybenzoyl-CoA reductase (4-HBCR) is a key enzyme in the anaerobic metabolism of phenolic compounds. It catalyzes the reductive removal of the hydroxyl group from the aromatic ring yielding benzoyl-CoA and water. The subunit architecture, amino acid sequence, and the cofactor/metal content indicate that it belongs to the xanthine oxidase (XO) family of molybdenum cofactor-containing enzymes. 4-HBCR is an unusual XO family member as it catalyzes the irreversible reduction of a CoA-thioester substrate. A radical mechanism has been proposed for the enzymatic removal of phenolic hydroxyl groups. In this work we studied the spectroscopic and electrochemical properties of 4-HBCR by EPR and Mossbauer spectroscopy and identified the pterin cofactor as molybdopterin mononucleotide. In addition to two different [2Fe-2S] clusters, one FAD and one molybdenum species per monomer, we also identified a [4Fe-4S] cluster/monomer, which is unique among members of the XO family. The reduced [4Fe-4S] cluster interacted magnetically with the Mo(V) species, suggesting that the centers are in close proximity, (<15 A apart). Additionally, reduction of the [4Fe-4S] cluster resulted in a loss of the EPR signals of the [2Fe-2S] clusters probably because of magnetic interactions between the Fe-S clusters as evidenced in power saturation studies. The Mo(V) EPR signals of 4-HBCR were typical for XO family members. Under steady-state conditions of substrate reduction, in the presence of excess dithionite, the [4Fe-4S] clusters were in the fully oxidized state while the [2Fe-2S] clusters remained reduced. The redox potentials of the redox cofactors were determined to be: [2Fe-2S](+1/+2) I, -205 mV; [2Fe-2S] (+1/+2) II, -255 mV; FAD/FADH( small middle dot)/FADH, -250 mV/-470 mV; [4Fe-4S](+1/+2), -465 mV and Mo(VI)/(V)/(VI), -380 mV/-500 mV. A catalytic cycle is proposed that takes into account the common properties of molybdenum cofactor enzymes and the special one-electron chemistry of dehydroxylation of phenolic compounds.

Kuper J, Palmer T, Mendel RR, Schwarz G
Mutations in the molybdenum cofactor biosynthetic protein Cnx1G from Arabidopsis thaliana define functions for molybdopterin binding, molybdenum insertion, and molybdenum cofactor stabilization.
Proc Natl Acad Sci U S A. 2000; 97: 6475-80
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The molybdenum cofactor (Moco), a highly conserved pterin compound coordinating molybdenum (Mo), is required for the enzymatic activities of molybdoenzymes. In all organisms studied so far Moco is synthesized by a unique and evolutionary old multistep pathway that requires the activities of at least six gene products. In eukaryotes, the last step of Moco synthesis, i.e., transfer and insertion of Mo into molybdopterin (MPT), is catalyzed by the two-domain proteins Cnx1 in plants and gephyrin in mammals. Both domains (E and G) of these proteins are able to bind MPT in vitro. Here, we show the identification and mutational dissection of functionally important regions within the Cnx1 G domain that are essential for MPT binding, the conversion of MPT to Moco, and Moco stabilization. By functional screening for mutants in the Cnx1 G domain that are no longer able to complement Escherichia coli mogA mutants, we found two classes of mutations in highly conserved amino acid residues. (i) The first class affects in vitro binding of MPT to the protein and the stabilization of Moco, the product of the G domain. (ii) The second class is represented by two independent mutations in the aspartate 515 position that is not affected in MPT binding and Moco stabilization; rather the conversion of MPT to Moco by using bound MPT and a yet unknown form of Mo is completely abolished. The results presented here provide biochemical evidence for a purified Cnx1 G domain catalyzing the insertion of Mo into MPT.

Lorite MJ, Tachil J, Sanjuan J, Meyer O, Bedmar EJ
Carbon monoxide dehydrogenase activity in Bradyrhizobium japonicum.
Appl Environ Microbiol. 2000; 66: 1871-6
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Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)(2) subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the B. japonicum CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein.

Xi H, Schneider BL, Reitzer L
Purine catabolism in Escherichia coli and function of xanthine dehydrogenase in purine salvage.
J Bacteriol. 2000; 182: 5332-41
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Escherichia coli is not known to utilize purines, other than adenine and adenosine, as nitrogen sources. We reinvestigated purine catabolism because a computer analysis suggested several potential sigma(54)-dependent promoters within a 23-gene cluster whose products have homology to purine catabolic enzymes. Our results did not provide conclusive evidence that the sigma(54)-dependent promoters are active. Nonetheless, our results suggest that some of the genes are metabolically significant. We found that even though several purines did not support growth as the sole nitrogen source, they did stimulate growth with aspartate as the nitrogen source. Cells produced (14)CO(2) from minimal medium containing [(14)C]adenine, which implies allantoin production. However, neither ammonia nor carbamoyl phosphate was produced, which implies that purine catabolism is incomplete and does not provide nitrogen during nitrogen-limited growth. We constructed strains with deletions of two genes whose products might catalyze the first reaction of purine catabolism. Deletion of one eliminated (14)CO(2) production from [(14)C]adenine, which implies that its product is necessary for xanthine dehydrogenase activity. We changed the name of this gene to xdhA. The xdhA mutant grew faster with aspartate as a nitrogen source. The mutant also exhibited sensitivity to adenine, which guanosine partially reversed. Adenine sensitivity has been previously associated with defective purine salvage resulting from impaired synthesis of guanine nucleotides from adenine. We propose that xanthine dehydrogenase contributes to this purine interconversion.

Thapper A, Lorber C, Fryxelius J, Behrens A, Nordlander E
Synthesis and reactivity studies of model complexes for molybdopterin-dependent enzymes.
J Inorg Biochem. 2000; 79: 67-74
Display abstract
The molybdenum cofactor (Moco)-containing enzymes are divided into three classes that are named after prototypical members of each family, viz. sulfite oxidase, DMSO reductase and xanthine oxidase. Functional or structural models have been prepared for these three prototypical enzymes: (i) The complex [MoO2(mnt)2]2- (mnt2- = 1,2-dicyanoethylenedithiolate) has been found to be able to oxidize hydrogen sulfite to HSO4- and is thus a functional model of sulfite oxidase. Kinetic and computational studies indicate that the reaction proceeds via attack of the substrate at one of the oxo ligands of the complex, rather than at the metal. (ii) The coordination geometries of the mono-oxo [Mo(VI)(O-Ser)(S2)2] entity (S2 = dithiolene moiety of molybdopterin) found in the crystal structure of R. sphaeroides DMSO reductase and the corresponding des-oxo Mo(IV) unit have been reproduced in the complexes [M(VI)O(OSiR3)(bdt)2] and [M(VI)O(OSiR3)(bdt)2] (M = Mo,W; bdt = benzene dithiolate). (iii) A facile route has been developed for the preparation of complexes containing a cis-Mo(VI)OS molybdenum oxo, sulfido moiety similar to that detected in the oxidized form of xanthine oxidase.

Gremer L, Kellner S, Dobbek H, Huber R, Meyer O
Binding of flavin adenine dinucleotide to molybdenum-containing carbon monoxide dehydrogenase from Oligotropha carboxidovorans. Structural and functional analysis of a carbon monoxide dehydrogenase species in which the native flavoprotein has been replaced by its recombinant counterpart produced in Escherichia coli.
J Biol Chem. 2000; 275: 1864-72
Display abstract
The carbon monoxide (CO) dehydrogenase of Oligotropha carboxidovorans is composed of an S-selanylcysteine-containing 88. 7-kDa molybdoprotein (L), a 17.8-kDa iron-sulfur protein (S), and a 30.2-kDa flavoprotein (M) in a (LMS)(2) subunit structure. The flavoprotein could be removed from CO dehydrogenase by dissociation with sodium dodecylsulfate. The resulting M(LS)(2)- or (LS)(2)-structured CO dehydrogenase species could be reconstituted with the recombinant apoflavoprotein produced in Escherichia coli. The formation of the heterotrimeric complex composed of the apoflavoprotein, the molybdoprotein, and the iron-sulfur protein involves structural changes that translate into the conversion of the apoflavoprotein from non-FAD binding to FAD binding. Binding of FAD to the reconstituted deflavo (LMS)(2) species occurred with second-order kinetics (k(+1) = 1350 M(-1) s(-1)) and high affinity (K(d) = 1.0 x 10(-9) M). The structure of the resulting flavo (LMS)(2) species at a 2.8-A resolution established the same fold and binding of the flavoprotein as in wild-type CO dehydrogenase, whereas the S-selanylcysteine 388 in the active-site loop on the molybdoprotein was disordered. In addition, the structural changes related to heterotrimeric complex formation or FAD binding were transmitted to the iron-sulfur protein and could be monitored by EPR. The type II 2Fe:2S center was identified in the N-terminal domain and the type I center in the C-terminal domain of the iron-sulfur protein.

Kang BS, Kim YM
Cloning and molecular characterization of the genes for carbon monoxide dehydrogenase and localization of molybdopterin, flavin adenine dinucleotide, and iron-sulfur centers in the enzyme of Hydrogenophaga pseudoflava.
J Bacteriol. 1999; 181: 5581-90
Display abstract
Carbon monoxide dehydrogenases (CO-DH) are the enzymes responsible for the oxidation of CO to carbon dioxide in carboxydobacteria and consist of three nonidentical subunits containing molybdopterin flavin adenine dinucleotide (FAD), and two different iron-sulfur clusters (O. Meyer, K. Frunzke, D. Gadkari, S. Jacobitz, I. Hugendieck, and M. Kraut, FEMS Microbiol. Rev. 87:253-260, 1990). The three structural genes of CO-DH in Hydrogenophaga pseudoflava were cloned and characterized. The genes were clustered on the chromosome in the transcriptional order cutM-cutS-cutL. The cloned cutM, cutS, and cutL genes had open reading frames of 864, 492, and 2,412 nucleotides, coding for proteins with calculated molecular weights of 30,694, 17,752, and 87,224, respectively. The overall identities in the nucleotide sequence of the genes and the amino acid sequence of the subunits with those of other carboxydobacteria were 64.5 to 74.3% and 62.8 to 72.3%, respectively. Primer extension analysis revealed that the transcriptional start site of the genes was the nucleotide G located 47 bp upstream of the cutM start codon. The deduced amino acid sequences of the three subunits of CO-DH implied the presence of molybdenum cofactor, FAD, and iron-sulfur centers in CutL, CutM, and CutS, respectively. Fluorometric analysis coupled with denaturing polyacrylamide gel electrophoresis of fractions from hydroxyapatite column chromatography in the presence of 8 M urea of active CO-DH and from gel filtration of spontaneously inactivated enzyme revealed that the large and medium subunits of CO-DH in H. pseudoflava bind molybdopterin and FAD cofactors, respectively. Iron-sulfur centers of the enzyme were identified to be present in the small subunit on the basis of the iron content in each subunit eluted from the denaturing polyacrylamide gels.

Dias JM et al.
Crystal structure of the first dissimilatory nitrate reductase at 1.9 A solved by MAD methods.
Structure. 1999; 7: 65-79
Display abstract
BACKGROUND: The periplasmic nitrate reductase (NAP) from the sulphate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is induced by growth on nitrate and catalyses the reduction of nitrate to nitrite for respiration. NAP is a molybdenum-containing enzyme with one bis-molybdopterin guanine dinucleotide (MGD) cofactor and one [4Fe-4S] cluster in a single polypeptide chain of 723 amino acid residues. To date, there is no crystal structure of a nitrate reductase. RESULTS: The first crystal structure of a dissimilatory (respiratory) nitrate reductase was determined at 1.9 A resolution by multiwavelength anomalous diffraction (MAD) methods. The structure is folded into four domains with an alpha/beta-type topology and all four domains are involved in cofactor binding. The [4Fe-4S] centre is located near the periphery of the molecule, whereas the MGD cofactor extends across the interior of the molecule interacting with residues from all four domains. The molybdenum atom is located at the bottom of a 15 A deep crevice, and is positioned 12 A from the [4Fe-4S] cluster. The structure of NAP reveals the details of the catalytic molybdenum site, which is coordinated to two MGD cofactors, Cys140, and a water/hydroxo ligand. A facile electron-transfer pathway through bonds connects the molybdenum and the [4Fe-4S] cluster. CONCLUSIONS: The polypeptide fold of NAP and the arrangement of the cofactors is related to that of Escherichia coli formate dehydrogenase (FDH) and distantly resembles dimethylsulphoxide reductase. The close structural homology of NAP and FDH shows how small changes in the vicinity of the molybdenum catalytic site are sufficient for the substrate specificity.

Ragsdale SW, Kumar M
Nickel-Containing Carbon Monoxide Dehydrogenase/Acetyl-CoA Synthase(,).
Chem Rev. 1996; 96: 2515-2540

Xiang Q, Edmondson DE
Purification and characterization of a prokaryotic xanthine dehydrogenase from Comamonas acidovorans.
Biochemistry. 1996; 35: 5441-50
Display abstract
Xanthine dehydrogenase (XDH) is induced in Comamonas acidovorans cells incubated in a limited medium with hypoxanthine as the only carbon and nitrogen source. The enzyme has been purified to homogeneity using standard techniques and characterized. It contains two subunits with M(r) values of 90 and 60 kDa. Gel filtration studies show the enzyme to have an alpha 2 beta 2 native structure. No precursor form of the enzyme is observed on Western blot analysis of cell extracts obtained at various stages of enzyme induction. Metal analysis of the purified enzyme shows 1.1 Mo, 4.0 Fe, and 3.6 phosphorus atoms per alpha beta protomer. Cofactor analysis shows the enzyme to contain a single molybdopterin mononucleotide and one FAD per alpha beta protomer. Electron spin resonance and circular dichroism spectral studies of the oxidized and reduced forms of the enzyme suggest the Fe centers to be two nonidentical [2Fe-2S] clusters. Electron spin resonance signals due to Mo(V) and neutral FAD radical are also observed in the reduced form of the enzyme. Purified enzyme preparations ranged from 70% to 100% functionality. The enzyme is irreversibly inactivated by CN- and is inhibited on incubation with allopurinol. With xanthine and NAD+ as substrates the enzyme has a specific activity of 50 units/mg, a kcat value of 120 s-1, an activity/flavin ratio of 1930, and respective Km values of 66 and 160 mM. Using 8-D-xanthine as substrate, a DV value of 1.8 is found with no change in Km. Thus, the Km and KD values of the enzyme for xanthine are equal. These data show Comamonas XDH to exhibit structural properties similar to bovine milk xanthine oxidase/dehydrogenase and to chicken liver xanthine dehydrogenase. Although the bacterial enzyme exhibits a 6-7-fold greater turnover rate than bovine or avian enzymes, the catalytic efficiencies (as measured by V/K) are similar for all three enzymes.

Huber R et al.
A structure-based catalytic mechanism for the xanthine oxidase family of molybdenum enzymes.
Proc Natl Acad Sci U S A. 1996; 93: 8846-51
Display abstract
The crystal structure of the xanthine oxidase-related molybdenum-iron protein aldehyde oxido-reductase from the sulfate reducing anaerobic Gram-negative bacterium Desulfovibrio gigas (Mop) was analyzed in its desulfo-, sulfo-, oxidized, reduced, and alcohol-bound forms at 1.8-A resolution. In the sulfo-form the molybdenum molybdopterin cytosine dinucleotide cofactor has a dithiolene-bound fac-[Mo, = O, = S, ---(OH2)] substructure. Bound inhibitory isopropanol in the inner compartment of the substrate binding tunnel is a model for the Michaelis complex of the reaction with aldehydes (H-C = O,-R). The reaction is proposed to proceed by transfer of the molybdenum-bound water molecule as OH- after proton transfer to Glu-869 to the carbonyl carbon of the substrate in concert with hydride transfer to the sulfido group to generate [MoIV, = O, -SH, ---(O-C = O, -R)). Dissociation of the carboxylic acid product may be facilitated by transient binding of Glu-869 to the molybdenum. The metal-bound water is replenished from a chain of internal water molecules. A second alcohol binding site in the spacious outer compartment may cause the strong substrate inhibition observed. This compartment is the putative binding site of large inhibitors of xanthine oxidase.

Chan MK, Mukund S, Kletzin A, Adams MW, Rees DC
Structure of a hyperthermophilic tungstopterin enzyme, aldehyde ferredoxin oxidoreductase.
Science. 1995; 267: 1463-9
Display abstract
The crystal structure of the tungsten-containing aldehyde ferredoxin oxidoreductase (AOR) from Pyrococcus furiosus, a hyperthermophilic archaeon (formerly archaebacterium) that grows optimally at 100 degrees C, has been determined at 2.3 angstrom resolution by means of multiple isomorphous replacement and multiple crystal form averaging. AOR consists of two identical subunits, each containing an Fe4S4 cluster and a molybdopterin-based tungsten cofactor that is analogous to the molybdenum cofactor found in a large class of oxotransferases. Whereas the general features of the tungsten coordination in this cofactor were consistent with a previously proposed structure, each AOR subunit unexpectedly contained two molybdopterin molecules that coordinate a tungsten by a total of four sulfur ligands, and the pterin system was modified by an intramolecular cyclization that generated a three-ringed structure. In comparison to other proteins, the hyperthermophilic enzyme AOR has a relatively small solvent-exposed surface area, and a relatively large number of both ion pairs and buried atoms. These properties may contribute to the extreme thermostability of this enzyme.

Hensgens CM, Hagen WR, Hansen TA
Purification and characterization of a benzylviologen-linked, tungsten-containing aldehyde oxidoreductase from Desulfovibrio gigas.
J Bacteriol. 1995; 177: 6195-200
Display abstract
Desulfovibrio gigas NCIMB 9332 cells grown in ethanol-containing medium with 0.1 microM tungstate contained a benzylviologen-linked aldehyde oxidoreductase. The enzyme was purified to electrophoretic homogeneity and found to be a homodimer with a subunit M(r) of 62,000. It contained 0.68 +/- 0.08 W, 4.8 Fe, and 3.2 +/- 0.2 labile S per subunit. After acid iodine oxidation of the purified enzyme, a fluorescence spectrum typical for form A of molybdopterin was obtained. Acetaldehyde, propionaldehyde, and benzaldehyde were excellent substrates, with apparent Km values of 12.5, 10.8, and 20 microM, respectively. The natural electron acceptor is not yet known; benzylviologen was used as an artificial electron acceptor (apparent Km, 0.55 mM). The enzyme was activated by potassium ions and strongly inhibited by cyanide, arsenite, and iodoacetate. In the as-isolated enzyme, electron paramagnetic resonance studies readily detected W(V) as a complex signal with g values in the range of 1.84 to 1.97. The dithionite-reduced enzyme exhibited a broad signal at low temperature with g = 2.04 and 1.92; this is indicative of a [4Fe-4S]1+ cluster interacting with a second paramagnet, possibly the S = 1 system of W(IV). Until now W-containing aldehyde oxidoreductases had only been found in two Clostridium strains and two hyperthermophilic archaea. The D. gigas enzyme is the first example of such an enzyme in a gram-negative bacterium.

Volbeda A, Charon MH, Piras C, Hatchikian EC, Frey M, Fontecilla-Camps JC
Crystal structure of the nickel-iron hydrogenase from Desulfovibrio gigas.
Nature. 1995; 373: 580-7
Display abstract
The X-ray structure of the heterodimeric Ni-Fe hydrogenase from Desulfovibrio gigas, the enzyme responsible for the metabolism of molecular hydrogen, has been solved at 2.85 A resolution. The active site, which appears to contain, besides nickel, a second metal ion, is buried in the 60K subunit. The 28K subunit, which coordinates one [3Fe-4S] and two [4Fe-4S] clusters, contains an amino-terminal domain with similarities to the redox protein flavodoxin. The structure suggests plausible electron and proton transfer pathways.

Hochheimer A, Schmitz RA, Thauer RK, Hedderich R
The tungsten formylmethanofuran dehydrogenase from Methanobacterium thermoautotrophicum contains sequence motifs characteristic for enzymes containing molybdopterin dinucleotide.
Eur J Biochem. 1995; 234: 910-20
Display abstract
Formylmethanofuran dehydrogenases are molybdenum or tungsten iron-sulfur proteins containing a pterin dinucleotide cofactor. We report here on the primary structures of the four subunits FwdABCD of the tungsten enzyme from Methanobacterium thermoautotrophicum which were determined by cloning and sequencing the encoding genes fwdABCD. FwdB was found to contain sequence motifs characteristic for molybdopterin-dinucleotide-containing enzymes indicating that this subunit harbors the active site. FwdA, FwdC and FwdD showed no significant sequence similarity to proteins in the data bases. Northern blot analysis revealed that the four fwd genes form a transcription unit together with three additional genes designated fwdE, fwdF and fwdG. A 17.8-kDa protein and an 8.6-kDa protein, both containing two [4Fe-4S] cluster binding motifs, were deduced from fwdE and fwdG. The open reading frame fwdF encodes a 38.6-kDa protein containing eight binding motifs for [4Fe-4S] clusters suggesting the gene product to be a novel polyferredoxin. All seven fwd genes were expressed in Escherichia coli yielding proteins of the expected size. The fwd operon was found to be located in a region of the M. thermoautotrophicum genome encoding molybdenum enzymes and proteins involved in molybdopterin biosynthesis.

Volk M, Meyer O, Frunzke K
Metabolic relationship between the CO dehydrogenase molybdenum cofactor and the excretion of urothione by Hydrogenophaga pseudoflava.
Eur J Biochem. 1994; 225: 1063-71
Display abstract
Urothione was isolated as an excretion product of Hydrogenophaga pseudoflava and other bacteria at amounts approaching 253 micrograms/l of culture corresponding to 44 micrograms/g bacterial dry mass. The compound was identified as urothione by co-chromatography with urothione isolated from human urine, its characteristic ultraviolet and visible absorption spectra, oxidation to pterin-6-carboxylic-7-sulfonic acid by alkaline permanganate, 1H-NMR spectroscopy, double-quantum-filtered Fourier-transform 1H correlated spectroscopy, circular-dichroism spectroscopy and mass spectroscopy. A metabolic relationship between urothione and the carbon monoxide dehydrogenase molybdenum cofactor was suggested by a 1.1:0.5 molar ratio between urothione excreted and degradation of carbon monoxide dehydrogenase, a coincidence of urothione excretion and induction of carbon monoxide dehydrogenase with different species of carboxidotrophic bacteria, a structural relationship between molybdopterin cytosine dinucleotide of the carbon monoxide dehydrogenase molybdenum cofactor and urothione, and the demonstrated conversion of the carbon monoxide dehydrogenase molybdenum cofactor to urothione in vitro. A pathway for the conversion of the H. pseudoflava carbon monoxide dehydrogenase molybdenum cofactor to urothione has been proposed which involves molybdopterin cytosine dinucleotide, molybdopterin, phospho-norurothione and norurothione.

Gladyshev VN, Khangulov SV, Axley MJ, Stadtman TC
Coordination of selenium to molybdenum in formate dehydrogenase H from Escherichia coli.
Proc Natl Acad Sci U S A. 1994; 91: 7708-11
Display abstract
Formate dehydrogenase H from Escherichia coli contains multiple redox centers, which include a molybdopterin cofactor, an iron-sulfur center, and a selenocysteine residue (SeCys-140 in the polypeptide chain) that is essential for catalytic activity. Here we show that addition of formate to the native enzyme induces a signal typical of Mo(V) species. This signal is detected by electron paramagnetic resonance (EPR) spectroscopy. Substitution of 77Se for natural isotope abundance Se leads to transformation of this signal, indicating a direct coordination of Se with Mo. Mutant enzyme with cysteine substituted at position 140 for the selenocysteine residue has decreased catalytic activity and exhibits a different EPR signal. Since determination of the Se content of wild-type enzyme indicates approximately 1 gram atom per mol, we conclude that it is the Se atom of the SeCys-140 residue in the protein that is coordinated directly with Mo. The amino acid sequence flanking the selenocysteine residue in formate dehydrogenase H is similar to a conserved sequence found in several other prokaryotic molybdopterin-dependent enzymes. In most of these other enzymes a cysteine residue, or in a few cases a serine or a selenocysteine residue, occurs in the position corresponding to SeCys-140 of formate dehydrogenase H. By analogy with formate dehydrogenase H in these other enzymes, at least one of the ligands to Mo should be provided by an amino acid residue of the protein. This ligand could be the Se of a selenocysteine residue, sulfur of a cysteine residue, or, in the case of a serine residue, oxygen.

Fischer B, Schmalle H, Dubler E, Viscontini M
Molybdenum-pterin complexes: a functional and structural model for the binding site in the enzyme dimethyl sulfoxide reductase.
Adv Exp Med Biol. 1993; 338: 369-72

Johnson JL et al.
Prenatal diagnosis of molybdenum cofactor deficiency by assay of sulphite oxidase activity in chorionic villus samples.
J Inherit Metab Dis. 1991; 14: 932-7
Display abstract
Molybdenum cofactor deficiency is characterized by the absence of sulphite oxidase, xanthine dehydrogenase and aldehyde oxidase, the three known enzymes in man that require the cofactor for their activity. Prenatal diagnosis of the deficiency may be performed by assay of sulphite oxidase activity in cultured amniocytes. However, the activity in amniocytes is low and large numbers of cells are required for reliable assessment. We show that sulphite oxidase is present at high levels in chorionic villi obtained at 10-14 weeks gestation and can be assayed directly in the biopsy sample without cell culture. This assay has been applied to two pregnancies at risk for molybdenum cofactor deficiency with successful diagnoses of an unaffected and an affected fetus.

Karrasch M, Borner G, Enssle M, Thauer RK
The molybdoenzyme formylmethanofuran dehydrogenase from Methanosarcina barkeri contains a pterin cofactor.
Eur J Biochem. 1990; 194: 367-72
Display abstract
Recently formylmethanofuran dehydrogenase from the archaebacterium Methanosarcina barkeri has been shown to be a novel molybdo-iron-sulfur protein. We report here that the enzyme contains one mol of a bound pterin cofactor/mol molybdenum, similar but not identical to the molybdopterin of milk xanthine oxidase. The two pterins, after oxidation with I2 at pH 2.5, showed identical fluorescence spectra and, after oxidation with permanganate at pH 13, yielded pterin 6-carboxylic acid. They differed, however, in their apparent molecular mass: the pterin of formylmethanofuran dehydrogenase was 400 Da larger than that of milk xanthine oxidase, a property also exhibited by the pterin cofactor of eubacterial molybdoenzymes. A homogeneous formylmethanofuran dehydrogenase preparation was used for these investigations. The enzyme, with a molecular mass of 220 kDa, contained 0.5-0.8 mol molybdenum, 0.6-0.9 mol pterin, 28 +/- 2 mol non-heme iron and 28 +/- 2 mol acid-labile sulfur/mol based on a protein determination with bicinchoninic acid. The specific activity was 175 mumol.min-1.mg-1 (kcat = 640 s-1) assayed with methylviologen (app. Km = 0.02 mM) as artificial electron acceptor. The apparent Km for formylmethanofuran was 0.02 mM.

Rajagopalan KV
Molybdopterin--problems and perspectives.
Biofactors. 1988; 1: 273-8
Display abstract
Structural studies on several derivatives of molybdopterin, the organic component of the molybdenum cofactor common to virtually all molybdoenzymes, have provided definitive evidence for the presence of a reduced pterin ring with a 6-alkyl side-chain containing an enedithiol and a phosphomonoester in active molybdopterin. The cryptic nature of the cofactor makes structure - function studies on the protein-bound prosthetic group extremely difficult. In this review some of the relevant properties of the pterin ring are presented, and the possible roles of molybdopterin in catalysis are discussed.

Johnson C, Beedham C, Stell JG
Reaction of 1-amino- and 1-chlorophthalazine with mammalian molybdenum hydroxylases in vitro.
Xenobiotica. 1987; 17: 17-24
Display abstract
1-Amino- and 1-chlorophthalazine were tested for possible substrate activity with partially purified rabbit-liver aldehyde oxidase and bovine-milk xanthine oxidase. 1-Chlorophthalazine was a more efficient substrate than the parent compound, phthalazine, with either aldehyde oxidase or xanthine oxidase. The oxidation product of 1-chlorophthalazine was identified as 4-chloro-1-(2H)-phthalazinone on the basis of chromatographic, infra-red and mass-spectral data. 1-Aminophthalazine was oxidized by aldehyde oxidase to 4-amino-1-(2H)-phthalazinone but was a competitive inhibitor of xanthine oxidase. Kinetic studies at different pH values indicated that, in each case, it is the unprotonated form of 1-aminophthalazine that reacts with the molybdenum hydroxylases.

Herve F, Berger JP, Soulier J
[Sulfite and xanthine oxidase deficiency: a diagnosis based on 2 simple tests].
Ann Pediatr (Paris). 1986; 33: 857-857

Ushio K, Ishizuka M, Kogushi M, Fukui S, Toraya T
Identification of a dephosphorylated oxidation product of the molybdenum cofactor as 2-(1,2-dihydroxyethyl)thieno[3,2-g]pterin.
Biochem Biophys Res Commun. 1986; 135: 256-61
Display abstract
A new method was developed for the synthesis of 2-(1,2-dihydroxyethyl)thieno[3,2-g]pterin and related 2-substituted thienopterins. A dephosphorylated fluorescent oxidation product of the molybdenum cofactor isolated from xanthine oxidase (EC 1.2.3.2) was identified as 2-(1,2-dihydroxyethyl)thieno[3,2-g]pterin by comparison of electronic and fluorescence spectra and TLC behaviors with those of the synthetic compound.

Kramer S, Hageman RV, Rajagopalan KV
In vitro reconstitution of nitrate reductase activity of the Neurospora crassa mutant nit-1: specific incorporation of molybdopterin.
Arch Biochem Biophys. 1984; 233: 821-9
Display abstract
The reduced, metal-free pterin of the molybdenum cofactor has been termed molybdopterin. Oxidation of any molybdopterin-containing protein in the presence or absence of iodine yields oxidized molybdopterin derivatives termed Form A and Form B, respectively. Application of these procedures to whole cells and cell extracts has demonstrated the presence of molybdopterin in wild-type Neurospora crassa, and its absence in the cofactor-deficient mutant nit-1. In order to demonstrate that the reconstitution of nitrate reductase activity in nit-1 extracts results from the incorporation of molybdopterin into the apoprotein, active molybdopterin, free of contaminating amino acids or peptides, was isolated from chicken liver sulfite oxidase and used in the reconstitution system. The results show that, during reconstitution, exogenous molybdopterin is specifically incorporated into the nitrate reductase protein, confirming the role of molybdopterin as the organic moiety of the molybdenum cofactor.

Meyer O, Rajagopalan KV
Selenite binding to carbon monoxide oxidase from Pseudomonas carboxydovorans. Selenium binds covalently to the protein and activates specifically the CO----methylene blue reaction.
J Biol Chem. 1984; 259: 5612-7
Display abstract
The CO----methylene blue and CO----dichlorophenol indophenol activities of carbon monoxide oxidase were specifically activated upon aerobic incubation with selenite, whereas the NADH----methylene blue activity was not altered. Fully active enzyme contained selenium, molybdenum, and flavin adenine dinucleotide in a 1:1:1 ratio. Selenium was covalently bound to the protein, probably between the sulfurs of half-cystine residues, and not a constituent of the molybdenum cofactor. The action of selenite was directed to the cytoplasmic species of carbon monoxide oxidase exclusively, whereas the CO----methylene blue activity of the membrane-bound enzyme remained unaffected.

Barber MJ, Siegel LM
Electron paramagnetic resonance and potentiometric studies of arsenite interaction with the molybdenum centers of xanthine oxidase, xanthine dehydrogenase, and aldehyde oxidase: a specific stabilization of the molybdenum(V) oxidation state.
Biochemistry. 1983; 22: 618-24

Ishizuka M, Ushio K, Toraya T, Fukui S
Formation of thieno[3,2-g]pterines from the molybdenum cofactor.
Biochem Biophys Res Commun. 1983; 111: 537-43
Display abstract
A fluorescent oxidation product of the molybdenum cofactor was isolated from Escherichia coli nitrate reductase (EC 1.9.6.1) and bovine milk xanthine oxidase (EC 1.2.3.2), which showed a visible absorption band at 395 nm and was dephosphorylated by alkaline phosphatase but not by phosphodiesterase I. The dephosphorylated species was oxidized by periodate to thieno[3,2-g]pterin-2-carbaldehyde which was quantitatively converted to thieno[3,2-g]pterin-2-carboxylic acid by subsequent treatment with Ag2O in 2 N NaOH. These results indicate that the oxidation product of the molybdenum cofactor is a thieno[3,2-g]pterin derivative with an unidentified side chain in the 2 position.

Johnson JL, Hainline BE, Rajagopalan KV
Characterization of the molybdenum cofactor of sulfite oxidase, xanthine, oxidase, and nitrate reductase. Identification of a pteridine as a structural component.
J Biol Chem. 1980; 255: 1783-6
Display abstract
The molybdenum cofactor has been isolated in an oxidized inactive form from purified molybdoenzymes. The isolated material is shown to be a novel pterin. The active cofactor is presumably composed of molybdenum and a reduced form of the pterin.

Mason R, Zubieta JA
Iron-sulfur proteins: structural chemistry of their chromophores and related systems.
Angew Chem Int Ed Engl. 1973; 12: 390-9

Hartenstein R
Characteristics of a partially purified aldehyde oxidase from the crayfish Cambarus bartoni.
Comp Biochem Physiol B. 1973; 45: 87-93

Sayles CD, Browder LW, Williamson JH
Expression of xanthine dehydrogenase activity during embryonic development of Drosophila melanogaster.
Dev Biol. 1973; 33: 213-7

Shinoda T, Glassman E
Multiple molecular forms of xanthine dehydrogenase and related enzymes. II. Conversion of one form of xanthine dehydrogenase to another by extracts of Drosophila melanogaster.
Biochim Biophys Acta. 1968; 160: 178-87

Smith ST, Rajagopalan KV, Handler P
Purification and properties of xanthine dehydroganase from Micrococcus lactilyticus.
J Biol Chem. 1967; 242: 4108-17

Keller EC Jr, Glassman E
Phenocopies of the ma-1 and ry mutants of Drosophila melanogaster: inhibition in vivo of xanthine dehydrogenase by 4-hydroxypyrazolo(3,4-d)pyrimidine.
Nature. 1965; 208: 202-3