Mononuclear Molybdenum-Containing Enzymesand/or selenium atoms, in a diversity of arrangements, that is the base of the classification of the mononuclear molybdoenzymes into three

Mononuclear Molybdenum-Containing EnzymesLuisa B Maia and José JG Moura, LAQV, REQUIMTE, FCT NOVA, Caparica, Portugal

© 2018 Elsevier Inc. All rights reserved.

Introduction 1The Xanthine Oxidase Family 2Introduction 2Enzymatic Machineries 3Mechanistic Strategies 5The Sulfite Oxidase Family 6Introduction 6Enzymatic Machineries 6Mechanistic Strategies 8The Dimethylsulfoxide Reductase Family 9Introduction 9Enzymatic Machineries 9Mechanistic Strategies 11Overview 13Acknowledgments 14References 14

Ch

AbbreviationsDMS DimethylsulfideDMSO DimethylsulfoxideDMSOR Dimethylsulfoxide reductaseFDH Formate dehydrogenase (all types of formate dehydrogenase enzymes)FDH-H Escherichia coli formate dehydrogenase H, from the formate–hydrogen lyase systemFDH-N E. coli formate dehydrogenase N, from the anaerobic nitrate–formate “respiratory” pathwayFDH-O E. coli formate dehydrogenase O, from the aerobic “respiratory” pathwaysFe/S Iron–sulfur center; mARC, mitochondrial amidoxime reducing componentMOSC From molybdenum cofactor sulfurase C-terminal domain (proteins involved in pyranopterin cofactor

biosynthesis)NaR Nitrate reductase (all types of nitrate reductase enzymes, prokaryotic and eukaryotic ones)NaRGHI “Respiratory” nitrate reductase (prokaryotic), after the name of the encoding genes, narG, H, and ISO Sulfite oxidaseXD Xanthine dehydrogenaseXO Xanthine oxidase

emistry Molecul

Introduction

Molybdenum is a metallic element (Fig. 1) essential to the great majority of organisms, from archaea and bacteria to higher plantsand mammals,1,2 being found in the active site of enzymes that catalyze (mostly) oxidation–reduction reactions that involve thetransfer of an oxygen or sulfur or hydrogen atom to/from a carbon, nitrogen, or sulfur atom of key metabolites.3–11

Apart from a very few exceptions, where the “orange protein”12–16 or the nitrogenase with its unique molybdenum/ironheteronuclear cofactor is included,17–29 all molybdoenzymes harbor one molybdenum ion coordinated by the cis-dithiolene(dSdC]CdSd) group of one or two pyranopterin cofactor molecules (Fig. 2A)3–11—the mononuclear molybdenum-containingenzymes that will be reviewed in this article. The coordination sphere of the molybdenum ion is completed by oxygen and/or sulfurand/or selenium atoms, in a diversity of arrangements, that is the base of the classification of the mononuclear molybdoenzymesinto three large families, denominated after one benchmark enzyme (Fig. 2B)3: the xanthine oxidase family, the sulfite oxidasefamily, and the dimethylsulfoxide reductase family.

Presently, more than 50 molybdoenzymes are known and the number of these metalloenzymes is increasing every year, withseveral more being foreseen to be identified in a near future, based on genomic analyses.2,30,31 The great majority of themolybdoenzymes are prokaryotic, with eukaryotes holding only a restricted number of molybdoenzymes (mammals, for examplehave only four).3–11 Over the last years, structural, spectroscopic, kinetic, and theoretical studies have provided an extraordinarily

ar Sciences and Chemical Engineering https://doi.org/10.1016/B978-0-12-409547-2.13932-0 1

https://doi.org/10.1016/B978-0-12-409547-2.13932-0

Mo Molybdenum

95.94

42

[Kr] 4d 5 5s1

Fig. 1 Molybdenum. This heavy metal, with atomic number 42 and an electronic configuration [Kr] 4d5 5s1, belongs to the “d-block” of the Periodic Table, group 6and period 5.

sulfite oxidasefamily

pyranopterin cofactor

Mo

xanthine oxidasefamily

X = S, Se, O, S-Cu-S(Cys)

dimethylsulfoxide reductasefamily

X,Y = O, S, Se, Asp, Ser,Cys, SeCys in variable coordinations

(A)

(B)

Fig. 2 Active site structure of three families of mononuclear molybdenum-containing enzymes. (A) The pyranopterin cofactor molecule is formed by pyrano(green)–pterin(blue)–dithiolene(red)–methylphosphate(black) moieties. The dithiolene (dSdC]CdSd) group forms a five-membered ene-1,2-dithiolate chelatering with the molybdenum ion. In eukaryotes, the cofactor is found in the simplest monophosphate form (R is a hydrogen atom), while in prokaryotes it is most oftenfound esterified with several nucleotides (R can be one cytidine monophosphate, guanosine monophosphate or adenosine monophosphate). (B) Structure of themolybdenum center of the three families. For simplicity, only the cis-dithiolene group of the pyranopterin cofactor is represented.

2 Mononuclear Molybdenum-Containing Enzymes

detailed picture of the mononuclear molybdenum-containing enzymes. Presently, several molybdoenzymes can be described withatomic and electronic details regarding their molybdenum active site centers, the other redox-active centers present and the overallstructure. Also their reaction mechanisms can be described with atomic and electronic details for each catalytic intermediary and keystructure–activity relationships have been established. This article provides an overview of the three families of mononuclear-molybdenum containing enzymes, focusing on the structural (“Enzymatic Machineries” sections) and mechanistic (“MechanisticStrategies” sections) aspects of the best-characterized members.

The Xanthine Oxidase Family

Introduction

The xanthine oxidase (XO) family comprises diverse molybdoenzymes, both from eukaryotes and prokaryotes, such as themammalian XO (Eq. 1) and aldehyde oxidase (Eq. 2) or the prokaryotic Desulfovibrio aldehyde oxidoreductases (Eq. 2), Eubacteriumbarkeri nicotinate dehydrogenase (Eq. 3), Pseudomonas putida quinoline 2-oxidoreductase (Eq. 4), Thauera aromatica4-hydroxybenzoyl-CoA reductase (Eq. 5), and Oligotropha carboxidovorans carbon monoxide dehydrogenase (Eq. 6).

Mononuclear Molybdenum-Containing Enzymes 3

ð1Þ

ð2Þ

ð3Þ

ð4Þ

ð5Þ

ð6Þ

Many of the enzymes from this family are involved in reactions of the carbon biogeochemical cycle and in “carbon-related” rolesin higher organisms.11 In fact, the primitive carbon cycle would have been dependent on molybdenum, as the metal (and tungsten)would have been essential for the earliest, strictly anaerobic, organisms to handle aldehydes and carboxylic acids, catalyzing theirinterconversion (aldehyde oxidoreductases).32 In higher organisms, aldehyde oxidase is responsible for the plant oxidation ofabscisic aldehyde to abscisic acid (a plant hormone involved in development processes and in a variety of abiotic and biotic stressresponses),33,34 the biosynthesis of indole-3-acetic acid (an auxin phytohormone),35 or the mammalian formation of retinoic acid(a metabolite of retinol (vitamin A) that is involved in growth and development).36–40 This family is also responsible for the purinecatabolism (hydroxylation of hypoxanthine and xanthine to urate by XO) in almost all forms of life41–49 (only a small number oforganisms (e.g., some yeasts) use other mechanisms to oxidize xanthine).50 Noteworthy, XO and aldehyde oxidase are also involvedin the mammalian metabolism of xenobiotic compounds, where they catalyze the hydroxylation of carbon centers of heterocyclicaromatic compounds and the oxidation of aldehydic groups; both enzymes have also been suggested to be involved in reactiveoxygen and nitrogen species-mediated signaling pathways and diseases.51–88

Enzymatic Machineries

The enzymes from the XO family are characterized by having an active site where (in its oxidized form) the molybdenum ion iscoordinated, in a square-pyramidal geometry, by an apical oxo group (Mo]O), two equatorial sulfur atoms from the cis-dithiolenegroup of one pyranopterin cofactor molecule, one equatorial catalytically labile –OH group (in most cases) and one equatorialterminal sulfo (Mo]S; in most cases) or seleno (Mo]Se) or oxo (Mo]O) group (Fig. 2).9,11,44–49,89 The mammalian XO andaldehyde oxidase,9,11,44–49,89 or the prokaryotic 4-hydroxybenzoyl-CoA reductase,90,91 harbor the characteristic molybdenumcenter, with a catalytically labile –OH group and an essential terminal sulfo group (Mo]S). Other enzymes, like the nicotinatedehydrogenase,92–94 have instead a terminal seleno group (Mo]Se), a group with a higher hydricity than the sulfo group, whatshould be essential for the nicotinate activation.95,96 The molybdenum-dependent oxidation of carbon dioxide (carbon monoxidedehydrogenase), on the contrary, requires the unique binuclear Mo/Cu cofactor, which is coordinated to the polypeptide chain byone cysteine residue through the Mo–S–Cu–S(Cys) motified.97–103

The bovine milk XO, the prototype enzyme of this family, is one of the best studied enzymes and its structure and function willbe here described in detail.

The mammalian XO is a cytoplasmatic enzyme that is synthesized as a NADþ-dependent dehydrogenase (denominatedxanthine dehydrogenase (XD), Eq. 7).44–49 Yet, the XD form can be rapidly converted into a “strict” oxidase form that reduces


dioxygen rather than NADþ—the commonly studied and very well documented XO (Eq. 8). This conversion can be reversible,

through oxidation of key cysteine residues, or irreversible, by limited proteolysis.47,104–125 However, the extent and rate of in vivoconversion of XD into XO have been a matter of great controversy.69,73,105,108,109,126–132

ð7Þ

ð8Þ

Besides the molybdenum center, the mammalian XO possesses three additional redox-active centers: two [2Fe–2S] centers andone FAD (Fig. 3). Structurally, the mammalian XO is a homodimeric enzyme (a2) with each monomer folded into three domains,that can be thought of as pseudo a0b0g0 subunits, each holding one type of redox-active center (the N-terminal domain holds theFe/S centers, the central domain contains the FAD site, and the C-terminal harbors the molybdenum center).116,133 The four redox-active centers are aligned in an almost linear fashion that defines an intramolecular electron transfer pathway delivering electronsfrom the molybdenum center to the FAD, the sites where the hydroxylation and dioxygen or NADþ reduction take place,respectively (with the Fe/S centers intermediating the electron transfer, as discussed below).

The other enzymes of the XO family share a high structural homology with the mammalian XO. The mammalian aldehydeoxidase holds one identical molybdenum center, as well as two Fe/S centers and one FAD center, in the same a2 structure. Thesame structural arrangement is observed in the carbon monoxide dehydrogenase, but in this case in a (abg)2 structure, with each

Fig. 3 Three-dimensional structure view and arrangement of redox-active cofactors of selected members of the XO family. Bovine XO, D. gigas aldehydeoxidoreductase plus flavodoxin, O. carboxidovorans carbon monoxide dehydrogenase, and T. aromatica hydroxybenzoyl-CoA reductase are represented.Top—Arrangement of the redox-active cofactors within the subunits. Bottom—Three-dimensional structure view of the proteins in the same orientation; the colorsof each subunit match the colors of the boxes in the top part. Only one subunit in XO and AOR (that are a2) or one abg group in the other two enzymes (that are(abg)2) is represented. The structures shown are based on the PDB files 1FO4 (XO), 1VLB (aldehyde oxidoreductase), 1FX1 (flavodoxin), 1N5W (carbon monoxidedehydrogenase), and 1RM6 (hydroxybenzoyl-CoA reductase).


redox-active center (one molybdenum, two Fe/S, and one FAD) being harbored by a distinct subunit (Fig. 3).97–103 The bacterialaldehyde oxidoreductase fromDesulfovibrio species is another remarkable example. In spite of having only two Fe/S centers (no FADcenter), this enzyme forms a complex with its physiological partner, a flavin-containing flavodoxin, (Eq. 9) that can be regarded asthe pseudo g0 subunit of structure of XO (Fig. 3).134–137 Other interesting structural arrangements can be found in the Escherichia coliperiplasmatic aldehyde oxidoreductase138,139 or the T. aromatica 4-hydroxybenzoyl-CoA reductase,90,91 two enzymes that displayconsiderable structural homology with XO, but possess an additional fifth redox-active center, one [4Fe–4S] center (in the FADsubunit), in a (abg)2 structure (Fig. 3).

ð9Þ

Mechanistic Strategies

XO family enzymes typically catalyze the hydroxylation of a CdH bond in aromatic heterocyclic compounds and inaldehydes.44–49,140 Such oxidative hydroxylations are seen in the XO-catalyzed conversion of xanthine to urate (Eq. 1), as well asin the reactions catalyzed by aldehyde oxidase and aldehyde oxidoreductase (Eq. 2), nicotinate dehydrogenase (Eq. 3), andquinoline 2-oxidoreductase (Eq. 4). There are, however, at least two important exceptions: the carbon monoxide dehydrogenase-catalyzed oxidation of carbon monoxide to carbon dioxide, which does not involve hydrolysis of a CdH bond (Eq. 6),97–103 andthe reaction of hydroxybenzoyl-CoA reductase, which involves the irreversible dehydroxylation of the phenol ring, an oxygen atomabstraction/reduction reaction (Eq. 5).141,142

The reaction mechanism of XO-catalyzed hydroxylation is presently well established (Fig. 4).44–49,116,133,143,144 Catalysis isinitiated with the activation of the molybdenum catalytically labile hydroxyl group (Mo–OH) by a neighboring conserveddeprotonated glutamate residue, to form an Mo6þ–O� core (base-assisted catalysis). The now deprotonated oxygen undertakesnucleophilic attack on the carbon atom to be hydroxylated, with the simultaneous transfer of hydride from substrate to the essentialsulfo group (Mo]S!ModSH), resulting in the formation of a covalent intermediate, Mo4þdOdCdR(dSH) (whereR represents the remainder of the substrate molecule). Water/hydroxide then displaces the hydroxylated product from themolybdenum coordination sphere to yield a Mo4þdOH(2)(dSH) core (enzyme reductive half-reaction). The two electronstransferred from the substrate to the molybdenum during the reductive half-reaction (Mo6þ!Mo4þ) are rapidly transferred, viathe Fe/S centers, to the FAD (Mo!Fe/S!FAD; Fig. 3), where the reduction of dioxygen (or NADþ, in the case of the XD form) takesplace (enzyme oxidative half-reaction). In the now oxidized molybdenum center (Mo4þ!Mo6þ), the sulfo group is deprotonatedand the initial Mo6þdOH(]S) core is regenerated. Intramolecular electron transfer within XO (Mo!Fe/S!FAD) is, therefore, anintegral aspect of the XO catalysis. In the XO family hydroxylation strategy two points must be highlighted: (i) water is the ultimatesource of the oxygen atom incorporated into the hydroxylated product (the molybdenum labile hydroxyl group (Mo–OH) thatends up in the product of the reaction (urate) is regenerated from a solvent water molecule when the catalytic cycle is closed)145,146;and (ii) dioxygen acts only as the oxidizing substrate. The hydroxylation reactions catalyzed by these molybdoenzymes is, in thisway, quite different from the reaction catalyzed by monooxygenases, as molybdoenzymes generate, rather than consume, reducingequivalents and use dioxygen as an oxidant, rather than as the source of the oxygen atom incorporated into product. The reactionmechanism of the hydroxybenzoyl-CoA reductase, involving the irreversible dehydroxylation of the phenol ring, that is, a reductionreaction of oxygen atom abstraction (Eq. 5) is believed to be the reverse of the XO one.141,142

2H++2e−

H2O

(Glu) -O− (Glu) -OH (Glu) -OH (Glu) -O−

(H+)

Fig. 4 Reaction mechanism of mammalian XO-catalyzed xanthine hydroxylation. See text for details.


The Sulfite Oxidase Family

Introduction

The sulfite oxidase (SO) family comprises eukaryotic and prokaryotic enzymes involved in sulfite oxidation (Eq. 10), namelydiverse prokaryotic sulfite dehydrogenases and plant, chicken, and mammalian SO. These enzymes are used by almost all forms oflife in the catabolism of sulfur-containing amino acids and other sulfur-containing compounds, oxidizing sulfite to sulfate.Certainly, sulfite oxidase is one of the most striking examples of the human dependence on molybdenum.147–152 Sulfite (derivednot only from the catabolism of sulfur-containing amino acids, but also from sulfur-containing xenobiotic compounds) is toxic andits controlled oxidation to sulfate is critical for survival. Underscoring this vital role, human sulfite oxidase deficiency results insevere neonatal neurological problems, including attenuated growth of the brain, mental retardation, seizures, and early death.Molybdenum-dependent sulfite-oxidizing enzymes are also important for some prokaryotes that are able to generate energy fromthe “respiratory” oxidation of inorganic sulfur compounds.153–156

The SO family enzymes are also involved in the nitrogen biogeochemical cycle, being responsible for the first and rate-limitingstep of eukaryotic assimilation of nitrate in plant, algae, and fungi (nitrate reductases; NaR; Eq. 11).157,158 (It should be noted thatthe eukaryotic assimilatory NaR is distinct from any type of prokaryotic NaR enzymes, which are classified as members of thedimethylsulfoxide reductase family (as described below).). In addition, this family includes also enzymes that catalyze otherreactions, such as the E. coli YedY or the mammalian mitochondrial amidoxime reducing component (mARC), enzymes that areinvolved in the reduction (dehydroxylation) of S- and N-hydroxylated compounds (Eq. 12). Also the MOSC protein family,involved in molybdenum center sulfuration, belongs to the SO family, as well as, other archaeal and bacterial enzymes of unknownfunction, identified on the basis of genomic analyses.

ð10Þ

ð11Þ

ð12Þ


The molybdenum center of the enzymes of the SO family is closely related to that of the XO family, but with the distinctive featureof having the polypeptide chain, through a cysteine residue, directly coordinated to the molybdenum. In these enzymes, themolybdenum center displays (in its oxidized form) the same square-pyramidal geometry, with the apical oxo group (Mo]O), butwith the equatorial plane formed by two sulfur atoms of the pyranopterin cofactor, one oxo group (Mo]O) and the cysteinethiolate ligand (ModS(Cys)) (Fig. 2).159–160

The chicken liver SO, the benchmark enzyme of this family, as well as the SO from humans and other vertebrates, is located inthe mitochondrial intermembrane space, where it catalyzes the oxidation of the toxic sulfite to sulfate, with the simultaneousreduction of cytochrome c (Eq. 13).161 These enzymes are homodimeric (a2), with each monomer folded into two domains, onecomprising one b-type heme (responsible for the cytochrome c reduction) and the other the molybdenum center (where the sulfiteoxidation takes place), both of which can be thought as pseudo a0b0 subunits (Fig. 5).162 Remarkably, the molybdenum and hemecenters of chicken SO have been found to be more than 30 Å apart in the crystal structure.162 Hence, during catalysis, aconformational alteration would have to take place, through the flexible polypeptide that links the two domains, to bring thetwo centers into sufficiently close proximity to allow the rapid intramolecular electron transfer observed, (sulfite)Mo !Fe(cytochrome).163–165

Contrary to vertebrate SO, the plant enzymes are peroxisomal and use (reduce) dioxygen as the physiological oxidant (Eq. 14).Most significantly, these enzymes hold only the molybdenum center (no heme) in a a2 structure that, nonetheless, shows the samebasic architecture and fold structure as the molybdenum domain of vertebrate SO (Fig. 5).166–169 The “nonuniformity” of sulfite-oxidizing enzymes continues with the prokaryotic counterparts (sulfite dehydrogenases): these are typically periplasmatic enzymesthat display different structural organizations and redox cofactor compositions, and whose physiological electron acceptor is mostoften a cytochrome.153–156,170–172 While most of the sulfite dehydrogenases appear to contain only the molybdenum center in ahomodimeric or monomeric organization,154,155,161,173 significantly different enzymes also exist. The sulfite dehydrogenase fromStarkeya novella is a heterodimeric (ab) enzyme, containing a c-type heme (in its b subunit) in addition to the molybdenum center(in the a subunit) (Fig. 5).174–176 However, contrary to the crystal structure of chicken SO, the two redox-active cofactors are in thiscase in close proximity to each other (16 Å). Moreover, while the basic structure of the molybdenum domain is maintained, theS. novella b subunit is found wrapped around the a subunit. In this way, in spite of not being covalently linked to the molybdenumdomain (as the heme domain of the chicken enzyme is), the S. novella folding ensures that the heme is “locked” in the “right place,”with no need of a conformational change to form a competent intramolecular electron transfer pathway. The S. novella enzyme isthus crystallized as a stable dimer, with the cofactors in the “correct positions,” in clear contrast to the chicken SO, with its mobile

Fig. 5 Three-dimensional structure view and arrangement of redox-active cofactors of selected members of the SO family. Chicken and plant (A. thaliana) sulfiteoxidase, S. novella sulfite dehydrogenase, plant (P. angusta) nitrate reductase and mammalian mARC are represented. Top—Arrangement of the redox-activecofactors within the subunits. Bottom—Three-dimensional structure view of the proteins in the same orientation; the colors of each subunit match the colors of theboxes in the top part. Only one subunit in chicken and plant SO and plant NaR (that are a2) is represented. In the case of plant NaR, only the 3D structure of thedomain holding the molybdenum center is represented (the remainder of the structure is not known yet). The three protein chain complex with mARC is representedonly schematically (its structure is not known yet). The structures shown are based on the PDB files 1SOX (chicken SO), 1OGP (A. thaliana SO), 2BPB (S. novellasulfite dehydrogenase), and 2BIH (NaR molybdenum domain).


molybdenum and heme domains that have to move relatively to each other during the catalytic cycle. Interestingly, the S. novella cheme is found in a position similar to that modeled for the b heme of chicken enzyme,176 thus suggesting that there is an idealmolybdenum/heme interaction mode that is reproduced in different forms of life, with different heme types and in differentphysiological contexts.

ð13Þ

ð14Þ

Eukaryotic assimilatory nitrate-reducing enzymes, that catalyze the nitrate reduction to nitrite with the simultaneous oxidationof NAD(P)H (Eq. 15), also belong to the SO family.48,177–185 These NaRs are cytoplasmatic homodimeric enzymes (a2), containing,besides the characteristic molybdenum center (responsible for the nitrate reduction), one b-type heme (involved in electrontransfer) and one FAD center (responsible for the NAD(P)H oxidation) (Fig. 5).177,186,187 Consistent with their classification asSO family members, the NaR molybdenum domain is strikingly similar to one of chicken and plant SO. The FAD domain has beensuggested to contribute to the correct positioning of the heme domain for intramolecular electron transfer. Interestingly (given thesimilarity with the sulfite-oxidizing enzymes), the phosphorylation of a serine residue in the linker region between the molybde-num and heme domains,188,189 followed by the binding of a specific regulatory protein, effectively inhibits the enzyme through theinhibition of the intramolecular electron transfer.190–193

ð15Þ

Another member of the SO family is the recently described fourth mammalian molybdoenzyme, mARC (after XO, AO, andSO).194 The physiological function of mARC is not yet known, but, because it can catalyze the reduction of N- and S-hydroxylatedcompounds (Eq. 12),195–198 mARC is probably involved in the detoxification of mutagenic and toxic aromatic hydroxyl-amines,such as N-hydroxylated DNA base derivates,199,200 among other possible roles.201–203 mARC is a monomeric mitochondrial


enzyme (outer membrane-bounded, facing the cytoplasm) and contains only the molybdenum center (with no additional redox-active centers).196,201,204,205 Yet, mARC functions in concert with cytochrome b5 and a NADH-dependent cytochrome b5 reductase,which are involved in electron transfer from the NADH molecules to the mARC (Fig. 5), thus forming a three-protein electrontransfer chain resembling the arrangement of redox-active cofactors seen in eukaryotic NaR enzymes: NAD(P)H!FAD!heme!Mo!hydroxylated compounds.194,195,204–207


SO family enzymes are proper oxotransferases that catalyze the “simple” transfer of one oxygen atom to, or from, a lone electronpair of the substrate, with water being the ultimate oxygen atom donor or acceptor, as is illustrated by the SO and NaR reactions.

Sulfite oxidation162,208–216 (Eq. 10; Fig. 6A) is initiated by the nucleophilic attack of the sulfite lone-pair of electrons on thecatalytically labile equatorial oxo group of the oxidized molybdenum center (Mo6þ]O), which can be thought as an electrophilicoxygen, resulting in the formation of a covalent reduced Mo4þdOdSO3equatorial intermediate.213,217–222 The ModO bond of thisintermediate is subsequently cleaved and the product (sulfate) replaced by a water molecule, leading to the formation of aMo4þdOH(2) core (the reductive half-reaction). In vertebrate SO, the two electrons transferred from the substrate to the molyb-denum are then intramolecularly transferred, one at a time, to the heme, where cytochrome c (the physiological partner) will bereduced, and the initial Mo6þ]O core is regenerated (oxidative half-reaction). Hence, in vertebrate SO, the two half-reactions arephysically separated, with one of the half-reactions being carried out at the molybdenum center and the other elsewhere (at theheme of SO); therefore, intramolecular electron transfer (Mo!heme) is an integral aspect of catalysis in vertebrate SO. On thecontrary, in plant SO, that is devoid of additional redox cofactors, the oxidative half-reaction also takes place at molybdenum center,with the one-electron reduction of dioxygen to superoxide radical anion, which, then, spontaneously dismutates to hydrogenperoxide.223

The reaction mechanism of NaR-catalyzed nitrate reduction to nitrite is believed to be the reverse of the SO one, with theabstraction of an oxygen atom from the nitrate molecule and the intramolecular electron flow occurring in the opposite direction(Eq. 11; Fig. 6B).179,182,185,186,224–227 Hence, catalysis is initiated with the reduction of the FAD center by NAD(P)H (the reductivehalf-reaction) and the two electrons being transferred, intramolecularly, through the heme, to the molybdenum center(FAD!heme!Mo). In the now reduced molybdenum center (Mo4þ), nitrate displaces the labile group and binds directly tothe molybdenum ion, resulting in the formation of a covalent reduced Mo4þdOdNO2 intermediate. The OdN bond of thisintermediate is subsequently cleaved, the product (nitrite) released and the Mo6þ]O core regenerated (the oxidative half-reaction).

The newly recognized members of this family clearly show that the chemistry carried out by the enzymes of the SO family ismore diverse than “just” the oxidation of sulfite or the reduction of nitrate. The recent identification of mammalian mARC andbacterial YcbX, YiiM or YedY, as well as of several MOSC proteins homologues (most of these not yet characterized), demonstratedthat the active site of SO family enzymes are also able to catalyze the reduction (dehydroxylation) of S- and N-hydroxylatedcompounds and sulfuration reactions.228–236

H++e−

H2O

H++e−

2H++2e−

H2O

(A)

(B)

Fig. 6 Reaction mechanism of vertebrate SO-catalyzed sulfite oxidation (A) and of plant NaR-catalyzed nitrate reductase (B). See text for details.


The Dimethylsulfoxide Reductase Family

Introduction

The dimethylsulfoxide reductase (DMSOR) family is probably the most diverse of the three families, comprising prokaryoticenzymes with remarkably different activities, including reduction of DMSO (Eq. 16), oxidation of arsenite (Eq. 17) and reduction ofarsenate (Eq. 18), reduction of nitrate (Eq. 11), and oxidation of nitrite (Eq. 19), oxidation of formate and reduction of carbondioxide (Eq. 20), reduction of polysulfide (Eq. 21), hydroxyl transfer within pyrogallol/phloroglucinol (Eq. 22) and even hydrationof acetylene (Eq. 23), among many other reactions. As such, the DMSOR family enzymes take part in many biogeochemical cycles.In the carbon cycle, for example, acetogens use formate dehydrogenases (FDH; Eq. 20) to fix carbon dioxide (reduce it) into formateand eventually form acetate, while other organisms use the same enzymes to catalyze the reverse reaction to derive energy and fulfillother metabolic roles. This is the case of E. coli, that expresses three FDHs: (i) a cytoplasmatic enzyme, designated formatedehydrogenase H (FDH-H), that is part of the formate–hydrogen lyase system and is involved in the oxidation of formate andgeneration of molecular hydrogen under fermentative (anaerobic) growth conditions237–239; (ii) a membrane-bound periplasm-facing FDH, designated formate dehydrogenase N (FDH-N), that is part of the anaerobic nitrate–formate “respiratory” pathway(catalyzed by a supermolecular formate:nitrate oxidoreductase system formed with a “respiratory” NaR) involved with thegeneration of a proton motive force240–247; and (iii) a second membrane-bound periplasm-faced FDH, designated formatedehydrogenase O (FDH-O), that operates under aerobic conditions in another nitrate–formate “respiratory” pathway.248–252

The DMSOR family enzymes are also involved in the nitrogen biogeochemical cycle, being responsible for the prokaryoticassimilatory and dissimilatory reduction of nitrate to nitrite (Eq. 11) and for the dissimilatory oxidation of nitrite to nitrate (Eq. 19).This family comprises three different types of NaR enzymes237,253–265: (i) dissimilatory “respiratory”membrane-bound cytoplasm-faced enzymes involved in the generation of a proton motive force across the cytoplasmatic membrane, (ii) dissimilatory periplas-matic enzymes that are suggested to be associated with the elimination of excess of reducing equivalents, and (iii) assimilatorycytoplasmatic enzymes responsible for nitrogen assimilation (enzymes distinct from the eukaryotic ones, that, as described above,belong to the SO family). The DMSOR family includes also the nitrite oxidoreductases (Eq. 19) from the nitrifiers. Thesemembrane-bound proteins, facing the periplasm266–273 or the cytoplasm,266,274–279 depending on the organism, have been poorlystudied, but there is evidence that they must share several structural features with the “respiratory” NaRs.280–282

ð16Þ

ð17Þ

ð18Þ

ð19Þ

ð20Þ

2� � þ þ 2� 2�
(Sn) þ 2e þ 2H ! (2H )S þ (Sn�1) (21)
ð22Þ

HdC�CdH þ H2O!H3CdCOH (23)


The enzymes from this family are characterized by having the molybdenum ion coordinated by four sulfur atoms of twopyranopterin cofactor molecules (Fig. 2). These molybdenum centers display a trigonal prismatic geometry, that is completed byoxygen and/or sulfur and/or selenium atoms in a diversity of arrangements, with the molybdenum ion being often found directlycoordinated by the polypeptide chain via aspartate, serine, cysteine, or selenocysteine residue side chains (in the oxidizedstate).9,11,44–49,89,283


Following the diversity in the reactions catalyzed, the DMSOR family is also the most diverse of the three families regardingsubunit composition and redox-active cofactors content (Fe/S centers, hemes and flavins).237,284,285 To restrict the informationpresented to a manageable size, only a limited number of enzymes will be here reviewed, namely DMSOR, NaR, and FDH enzymes.

The Rhodobacter sphaeroides DMSOR, the benchmark enzyme of this family is a periplasmatic monomeric protein, containingonly the molybdenum center as a redox-active group, with the molybdenum ion coordinated (in its oxidized form) by thecharacteristic two pyranopterin molecules plus one catalytically labile oxo group (Mo]O) and one serine side chain oxygenatom (ModO(Ser)) (Fig. 7).286–289 Contrary to the “simple” enzyme from Rhodobacter, the “respiratory” DMSOR from E. coli is amembrane-bound periplasm-facing heterotrimeric (abg) enzyme, that is comprised by (no crystal structure available yet)290–294:(i) a periplasmatic DMSO-reducing subunit that holds the molybdenum center and one [4Fe–4S] center; the amino acid residuesequence of the molybdenum-binding domain of this subunit is similar to the corresponding part of the Rhodobacter enzyme, withthe small domain containing the Fe/S center inserted in an N-terminal domain of the subunit; (ii) a second periplasmatic subunit,containing four [4Fe–4S] centers, that is thought to be involved in intramolecular electron transfer (from the membrane mena-quinol to the DMSO-reducing molybdenum center); and (iii) a membrane-integral subunit that has no redox-active cofactors andthat, presumably, binds the menaquinol necessary for enzyme reduction. Noteworthy, the “respiratory” DMSOR, that catalyzes anoxygen atom transfer reaction (Eq. 16), is remarkably similar to the “respiratory” polysulfide reductase, an enzyme involved in thereduction of inorganic sulfur to sulfide, a sulfur atom transfer reaction (Eq. 21). The Thermus thermophilus polysulfide reductase is amembrane-bound periplasm-faced enzyme, holding a similar subunit organization and cofactor composition to the E. coli“respiratory”DMSOR, but organized as a dimer of trimers ((abg)2).

240,295,296 The molybdenum center of the polysulfide reductase,however, is coordinated by a cysteine residue (Mo–S(Cys)) and probably by a catalytically labile hydroxyl group (Mo–OH).

Also the NaR enzymes display “simple” and “complex” subunit organizations and cofactor compositions to fulfill differentbiological roles, in different subcellular locations. The “respiratory”NaR from E. coli (product of the narG,H, and I genes and namedNaRGHI) is a “complex” dimer of heterotrimers, (abg)2, made up of (Fig. 7)257,297: (i) a cytoplasmatic nitrate-reducing NaRGsubunit that holds the molybdenum center and one [4Fe–4S] center; (ii) an electron transfer NaRH subunit that holds one [3Fe–4S]and three [4Fe–4S] centers, responsible for the intramolecular electron transfer from the membrane quinol pool to the nitrate-reducing molybdenum center; and (iii) a membrane-bound quinol-oxidizing NaRI subunit that holds two b-type hemes. On theother hand, the periplasmatic NaR fromDesulfovibrio desulfuricans (product of the napA gene) is a “simple”monomeric enzyme withonly one [4Fe–4S] center in addition to themolybdenum center (Fig. 7),298,299 while the enzyme from Cupriavidus necator (napA andnapB genes) is a dimer harboring two hemes in addition to the molybdenum center.300 Interestingly, although all these prokaryoticNaRs catalyze the two-electron reduction of nitrate to nitrite (Eq. 11), their molybdenum coordination sphere displays significant

Fig. 7 Three-dimensional structure view and arrangement of redox-active cofactors of selected members of the DMSOR family. R. sphaeroides dimethylsulfoxidereductase, D. desulfuricans periplasmatic nitrate reductase, E. coli “respiratory” nitrate reductase, E. coli formate dehydrogenase H, and E. coli formatedehydrogenase N are represented. Top—Arrangement of the redox-active cofactors within the subunits. Bottom—Three-dimensional structure view of the proteinsin the same orientation; the colors of each subunit match the colors of the boxes in the top part. Only one abg group of the E. coli respiratory NaR (that is (abg)2) andE. coli FDH N (that is (abg)3) is represented. The structures shown are based on the PDB files 1EU1 (DMSOR), 2NAP (D. desulfuricans periplasmatic NaR), 1Q16(E. coli respiratory NaR), 2IV2 (E. coli FDH H), and 1KQF (E. coli FDH N).


differences (apart from the characteristic common two pyranopterin cofactor molecules (Fig. 2)). In the “respiratory” membrane-bound NaR, the molybdenum ion is coordinated by an aspartate residue in a bidentate fashion (supposedly in its oxidized form), orby one oxygen atom from the aspartate residue coordinated in a monodentate mode plus one terminal oxo group (in the reducedform).246, 286 Yet, in the periplasmatic NaR from D. desulfuricans or C. necator, the molybdenum ion is coordinated by a cysteinesulfur atom plus one terminal sulfo group, forming a partial disulfide bond within each other.298–301 The E. coli and R. sphaeroidesperiplasmatic NaR, on their turn, complete the molybdenum coordination with the cysteine sulfur atom plus a terminal hydroxylgroup.302,303 The cytoplasmatic assimilatory NaR was not yet thoroughly studied, but its molybdenum ion is believed to becoordinated by a cysteine residue.304

As with the other enzymes, also the prokaryotic FDH enzymes (Eq. 20) are involved in diverse biochemical pathways and, thus,display different subunit composition and redox-active cofactors compositions.237,305–307 The E. coli FDH-H is a “simple” mono-meric enzyme, harboring only one [4Fe–4S] center (responsible for the intramolecular electron transfer to the physiologicalacceptor, probably a ferredoxin protein), in addition to the molybdenum center (Fig. 7).308–311 Its molybdenum ion is coordinatedby the characteristic two pyranopterin cofactor molecules plus a conserved essential selenocysteine residue (ModSe(Cys)) and aterminal sulfur atom (Mo]S).311,312 The E. coli FDH-N, on the contrary, is a “complex” trimer of trimers ((abg)3), with eight redox-active cofactors in a overall organization of the abg unit that is quite similar to that seen in the “respiratory” NaRGHI(Fig. 7)243,244,313–316: (i) a periplasmatic formate-oxidizing subunit that holds one molybdenum center and one [4Fe–4S]center, with the domains involved in binding the Fe/S and molybdenum centers structurally similar to the FDH-H monomer;(ii) a periplasmatic electron transfer subunit that harbors four [4Fe–4S] centers and is responsible for the intramolecular electrontransfer from the formate-oxidizing molybdenum center to the membrane quinone pool; and (iii) a membrane-bound quinone-reducing subunit that has two b-type hemes. The trimer of abg units, (abg)3, is very tightly packed and a cardiolipin molecule ismaintained at the trimer interface, thus suggesting that this complex arrangement is physiologically meaningful. The molybdenumof FDH-N is coordinated by selenocysteine and a terminal sulfur atom, as seen in its FDH-H counterpart.317 The FDH fromD. desulfuricans is also a “complex” periplasmatic enzyme, with a heterotrimeric structure (abg), but possesses only two [4Fe–4S]centers (on in each the a and b subunit) with four c hemes (g subunit); its molybdenum ion is by a selenocysteine and, probably, aterminal sulfur (no crystal structure is yet available yet).318,319 The enzymes from Ralstonia eutropha320–322 and Rhodobactercapsulatus323 are even more complex, holding seven Fe/S centers plus one FMN per abg protomer, in addition to the molybdenumcenter, which, in these two enzymes, is coordinated by a cysteine residue and a terminal sulfur atom (no crystal structure is as yetavailable).


The DMSOR family is catalytically extremely versatile,240,285,324,325 catalyzing reactions of oxygen atom transfer, as well exemplifiedby DMSOR (Eq. 16) or NaR (Eq. 11), but also of sulfur atom transfer, as is the case of the polysulfide reductase-catalyzed inorganicsulfur reduction to sulfide (Eq. 21), and of hydrogen atom transfer, as seen in the CdHbond cleavage/formation catalyzed by FDHs(Eq. 20), and even hydration reactions, as observed in the acetylene hydratase-catalyzed hydration of acetylene to acetaldehyde, anonredox reaction (Eq. 23).

Following the description in the “Enzymatic machineries” session, only the DMSOR, NaR, and FDH reaction mechanisms willbe here reviewed.

The reaction mechanism of DMSOR is presently well established (Fig. 8A).326–329 Catalysis is initiated with enzyme reduction bythe penta-heme DorC protein (the reductive half-reaction). Upon reduction of themolybdenum, the catalytically labile oxo group isprotonated and released in the form of a water molecule, resulting in the formation of a penta-coordinate, square-pyramidal center,with the serine residue occupying the apical position ((Ser)OdMo6þ]O!(Ser)OdMo4þ), via a (Ser)OdMo5þdOH interme-diate. The DMSO binding to the molybdenum yields a hexa-coordinate (Ser)OdMo4þdOdS(dimethyl) intermediate, whoseOdS bond cleavage leads to the product (DMS) release and regenerates the initial (Ser)OdMo6þ]O core (the oxidative half-reaction).

A similar mechanism has been suggested for the periplasmatic NaR from E. coli, but with nitrate binding to the Mo5þ oxidationstate (rather than Mo4þ).303 Hence, the catalytic cycle is, in this case, initiated with the molybdenum reduction by one electron, toform in a penta-coordinated desoxo Mo5þ center ((Cys)SdMo6þdOH!(Cys)SdMo5þ). Nitrate binds to this complex, throughthe oxygen atom to be abstracted, to form the hexa-coordinated intermediate with bound substrate. After reduction by the secondelectron, the OdN bond is cleaved, the product (nitrite) is released and the initial hexa-coordinated mono-oxo Mo6þ coreregenerated upon protonation. A comparable mechanistic strategy has been proposed for the sulfur atom transfer reaction of thepolysulfide reductase, with the terminal sulfur atom of the polysulfide displacing the catalytically labile hydroxyl group to becomedirectly coordinated to the reduced molybdenum atom ((Cys)SdMo6þdOH!(Cys)SdMo4þdS(polysulfide)).330,331

The NaRs lacking an exchangeable oxygen ligand, however, must follow a different mechanistic strategy. This is the case of theD. desulfuricans and C. necator periplasmatic NaR, which have a terminal sulfur atom, forming a partial disulfide bond with thesulfur atom of the cysteine residue side chain. Starting from the principle that the sulfo ligand would have to remain bound to themolybdenum throughout the catalytic cycle (i.e., that a sulfo ligand cannot be released as an oxo/hydroxyl group can), it has beenproposed, based on theoretical calculations, that the coordinating cysteine residue dissociates from the molybdenum atom througha “sulfur shift” mechanism (Fig. 8B).332,333 According to this mechanistic proposal, catalysis is initiated when nitrate reaches the(inactive) oxidized hexa-coordinated molybdenum center ((Cys)SdMo]S). The repulsive environment generated would triggerthe insertion of the sulfur atom into the (Cys)SdMo bond, to yield an active (Cys)SdSdMo core. As a result of this “sulfur shift,”

H++e−

H2O

H++e−

(A)

(B)

(C)

2H++2e−

H2O

inactive form

2H++2e−

2H++2e−

Fig. 8 Reaction mechanism of DMSO-catalyzed DMSO reduction (A), NaR-catalyzed nitrate reductase (B), and FDH-catalyzed formate oxidation and carbon dioxidereduction (C). See text for details. The reaction mechanism of FDH is exemplified with a selenocysteine-containing enzyme; a similar mechanism occurs in thecysteine-containing FDHs.


the molybdenum is formally reduced to Mo4þ and a new binding position is created to bind nitrate through one of its oxygen atomsand yield a hexa-coordinated (Cys)SdSdModONO2 intermediate. The cleavage of the OdNO2 bond leads to product release,leaving a hexa-coordinated mono-oxo Mo6þ core, with a “fresh” oxo group. This center can then be reduced and, after displacementof the oxo labile group by new molecule of nitrate, a new catalytic cycle can start.

A different mechanistic strategy was recently suggested for FDH, whose reactions do not involve oxygen atom exchange, but,instead, hydrogen atom transfer (Eq. 20).307,334–338 Formate oxidation catalysis (Fig. 8C, black arrows) is initiated with formatebinding to the oxidized molybdenum center, but not to the molybdenum ion directly. Instead, formate binds in a binding pocket,where conserved arginine and histidine residues anchor the molecule, through hydrogen bonds, forcing its Ca hydrogen to pointtoward the molybdenum terminal sulfo group (Mo6þ]S). Formate oxidation, then, proceeds through direct hydride transfer, fromthe formate molecule to the sulfo group, leading to the formation of a carbon dioxide moiety and a reduced molybdenum center


holding a protonated sulfo group (Mo4þdSH) (oxidative half-reaction). The two electrons transferred from formate to themolybdenum are, then, intramolecularly transferred to the respective FDH physiological partner and the initial Mo6þ]S centeris regenerated (reductive half-reaction). The carbon dioxide reduction follows the reverse mechanism, with the terminal protonatedsulfo group of the reduced molybdenum center (Mo4þdSH) acting as the hydride donor (Fig. 8C, blue arrows).337,338 According tothis “hydride transfer” mechanism, the FDH active site selenocysteine/cysteine residue remains bound to the molybdenum atomthroughout the all catalytic cycle. Yet, the presence of a hexa-coordinated molybdenum center is not mandatory for FDH catalysisand the formate oxidation/carbon dioxide reduction and can also proceed in a penta-coordinated center (as is the case in the otherenzymes described in this section).

Overview

Living organisms use molybdenum (mostly) to catalyze oxidation–reduction reactions that involve the transfer of an oxygen orsulfur or hydrogen atom to/from a carbon, nitrogen, or sulfur atom of key metabolites. Molybdenum has the adequate chemicalproperties to do this Redox Biochemistry, being redox-active under physiological conditions, with oxidation states ranging from 6þ,5þ to 4þ. This versatility allows the molybdoenzymes to catalyze either two-electron (Mo6þ$Mo4þ) or one-electron(Mo6þ$Mo5þ, Mo5þ$Mo4þ) oxidation–reduction reactions and, thus, couple obligatory two-electron and one-electron systems.This redox feature is key, for example, to the physiological coupling between the two-electron reduction of a “respiratory” substrateand a one-electron transfer protein. Also crucial to this coupling is the remainder content the molybdoenzymes. These enzymesevolved to harbor diverse redox-active cofactors (or no other cofactors at all) that allow the molybdoenzymes to interact withdiverse physiological redox partners, such as cytoplasmatic and periplasmatic cytochromes, ferredoxins, NAD(P), coenzyme F420 ormembrane quinols. As a consequence of the existence of several redox-active cofactors, the oxidative and reductive half-reactions ofthe catalytic cycle of the molybdoenzymes are, in many cases, physically separated, with one half-reaction occurring at themolybdenum center and the other taking place in another redox-active center. Consequently, intramolecular electron transfer isalso an integral aspect of catalysis in many molybdoenzymes (except in the case of the molybdenum-only enzymes).

Also key to its Redox Biochemistry, is the molybdenum very versatile first coordination sphere and its ability to form oxides andsulfides. In particular, the strong tendency of molybdenum to bind oxo groups, balanced by its ability to easily lose a single oxygenatom—a property that makes molybdenum complexes excellent “oxygen atom exchangers”—has been explored by organisms tocatalyze different oxotransfer reactions. In these, the molybdenum ion intermediates the transfer of an oxygen atom from water toproduct—oxygen atom insertion—or from substrate to water—oxygen atom abstraction (the “oxo transfer hypothesis” coined by Holmand others in the 1980s).

Hence, to catalyze an oxygen atom abstraction reaction (reduction), the molybdoenzyme needs a reduced penta-coordinatedmolybdenum center, with an available coordination position to bind the substrate directly to the molybdenum ion, via one oxygenatom. To catalyze an oxygen atom insertion reaction (oxidation), the molybdoenzyme needs an oxidized hexa-coordinatedmolybdenum center, with an available oxo/hydroxy group, which will be inserted into the product of the reaction. In either oxygenabstraction or insertion, during turnover (re-reduction or re-oxidation of the molybdenum center, respectively), the oxygen atomabstracted or inserted is removed or renovated, respectively, with a water molecule. On the contrary, to catalyze a hydrogen atomtransfer reaction, the molybdoenzymes use a terminal hydride donor/acceptor group present in the first coordination sphere of themolybdenum, to which the substrate binds or interacts with (no direct substrate binding to molybdenum). In the case of themolybdoenzymes, the hydride exchanger group is a terminal sulfo group (or seleno group), whose protonation state (pKa) is linkedto the oxidation state of the metal, in a way that an oxidized center holds its ligand deprotonated (Mo6þ]S/Se), while a reducedcenter has its ligand protonated (Mo4þdSH/SeH). In this way, hydrogen atom abstraction reactions use an oxidized molybdenumcenter, with a deprotonated sulfo group, while hydrogen atom insertion reactions use a reduced molybdenum center, with aprotonated sulfo group. In both hydrogen abstraction or insertion, during turnover (re-oxidation or re-reduction of the molybde-num center, respectively), the hydrogen atom abstracted or inserted is removed or renovated, respectively, in an “automatic”way, asis determined by the pKa of the molybdenum sulfo ligand.

These general mechanistic strategies have determined the composition of the molybdoenzymes active site. The SO familyenzymes are (in general) proper oxotransferases, that catalyze the “simple” transfer of one oxygen atom to, or from, a lone electronpair of the substrate (as is illustrated by the SO (Eq. 10) and NaR (Eq. 11) reactions). In accordance, their active site is a penta-coordinated molybdenum center, with no terminal sulfo group. The XO family enzymes can catalyze the two types of reaction in thesame catalytic cycle, oxygen and hydrogen atom transfer (as is exemplified by the XO reaction (Eq. 1)), and their active sites hold aterminal sulfo or seleno group in a penta-coordinated molybdenum center. The DMSOR family comprises enzymes with remark-ably different activities and their active sites are “tailor-made” to accomplish each function, from “simple” oxygen atom transfer(DMSOR reaction (Eq. 16)), in hexa-/penta-coordinated centers with no terminal sulfo group, to hydrogen atom transfer (FDHreaction (Eq. 20)), in a hexa-/penta-coordinated center, but with a terminal sulfo group.

Presently, several structure–activity relationships have already been identified in the mononuclear molybdenum-containingenzymes and it is clear that variations on common themes (“Lego pieces”) are combined to confer a determined structure andreactivity. Yet, almost certainly, new structure–activity relationships will come to light in the near future, stimulated by the discoveryof new molybdoenzymes and deeper studies.


Acknowledgments

We acknowledge Fundação para a Ciência e Tecnologia (FCT/MCTES) for the financial support granted to the Associate Laboratoryfor Green Chemistry—LAQV, which is financed by national funds from FCT/MCTES (UID/QUI/50006/2013) and co-financed bythe ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER—007265). LBM also thanks to FCT/MCTES for thefellowship grant (SFRH/BPD/111404/2015, which is financed by national funds and co-financed by FSE).

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Mononuclear Molybdenum-Containing Enzymesand/or selenium atoms, in a diversity of arrangements, that is the base of the classification of the mononuclear molybdoenzymes into three