Crystal Structure and Mechanism of CO Dehydrogenase, a Molybdo Iron-Sulfur Flavoprotein Containing S-Selanylcysteine

Home , Carbonyl selenide, Nicotine dehydrogenase

Proc. Natl. Acad. Sci. USA Vol. 96, pp. 8884–8889, August 1999 Biochemistry

Crystal structure and mechanism of CO dehydrogenase, a molybdo iron-sulfur flavoprotein containing S-selanylcysteine

HOLGER DOBBEK*†,LOTHAR GREMER‡,ORTWIN MEYER‡, AND ROBERT HUBER*

*Max-Planck-Institut fu¨r Biochemie, Abteilung Strukturforschung, D-82152 Martinsried, Germany; and ‡Lehrstuhl fu¨r Mikrobiologie, Universita¨t Bayreuth, D-95440 Bayreuth, Germany

Contributed by Robert Huber, May 27, 1999

ABSTRACT CO dehydrogenase from the aerobic bacte- requires conformational changes of M introduced through the rium Oligotropha carboxidovorans catalyzes the oxidation of binding of M to LS (unpublished data). The S subunit harbors ؉ CO with H2O, yielding CO2, two electrons, and two H . Its two [2Fe–2S] centers, which are proximal and distal, respec- crystal structure in the air-oxidized form has been determined tively, to the molybdenum center. They are traditionally des- to 2.2 Å. The active site of the enzyme, which contains ignated type I and II according to spectroscopic properties, but molybdenum with three oxygen ligands, molybdopterin- the assignment is controversial (8, 9). Recent experiments with cytosine dinucleotide and S-selanylcysteine, delivers the elec- M subunit-depleted CO dehydrogenase preparations assign trons to an intramolecular electron transport chain composed the proximal center to type I and the distal to type II of two types of [2Fe–2S] clusters and flavin-adenine dinucle- (unpublished data). otide. CO dehydrogenase is composed of an 88.7-kDa molyb- CO dehydrogenase has been grouped into the sequence doprotein (L), a 30.2-kDa flavoprotein (M), and a 17.8-kDa family of molybdenum hydroxylases (10). Other prominent iron-sulfur protein (S). It is organized as a dimer of LMS representatives of that family catalyze the oxidative hydroxy- heterotrimers and resembles xanthine dehydrogenase/oxidase lation of a diverse range of aldehydes and aromatic heterocy- in many, but not all, aspects. A mechanism based on a cles (9). The high-resolution structure of aldehyde oxidoreduc- structure with the bound suicide-substrate cyanide is sug- tase (Mop) from Desulfovibrio gigas has been solved and has gested and displays the necessity of S-selanylcysteine for the provided an insight into an active site of a molybdenum catalyzed reaction. hydroxylase (11). Mop and CO dehydrogenase both contain the MCD–molybdenum cofactor and the same [2Fe–2S] clus- CO is a trace gas and important scavenger for hydroxyl radicals ters. However, Mop is devoid of flavin and contains a single in the atmosphere. About one fifth of CO [115–230 Tg⅐yrϪ1 polypeptide chain, whereas CO dehydrogenase is a heterotri- (1)] is used by soil microorganisms (2, 3). CO has a short meric flavoprotein. lifetime within the soil, and consumption of atmospheric CO The CO dehydrogenase structural genes (cox)ofO. carbox- occurs mainly in the top few centimeters of the humus layer (O idovorans are clustered in the transcriptional order 5Ј-coxM- horizon), most likely under aerobic conditions. coxS-coxL-3Ј along with the Calvin cycle (cbb) and hydroge- Under aerobic conditions, CO can be used as the sole source nase (hox) genes on a 30-kb DNA segment of the 128-kilobase of carbon and energy by the carboxidotrophic bacteria, which megaplasmid pHCG3 (10, 12). CO dehydrogenase shows comprise a taxonomically diverse group of obligate or facul- significant sequence similarities to the two domains of Mop tative chemolithoautotrophic species (3, 4). Under anaerobic (10, 11). CoxL is 57% similar (25% identical) to amino acids conditions, CO is used with low affinity by a number of 175–907 of Mop, and CoxS is 74% similar (41% identical) to methanogenic archaea and acetogenic, sulfate-reducing, or amino acids 1–166 of Mop. phototrophic bacteria (3, 5). Generally, anaerobic CO dehydrogenases are nickel-containing [4Fe-4S] proteins. CO dehy- MATERIALS AND METHODS drogenases of methanogens or acetogens have acetyl-CoA synthetase activity. CO dehydrogenase (EC 1.2.99.2) of the Protein Crystallization. The protein was crystallized under carboxidotrophic bacterium Oligotropha carboxidovorans is a vapor diffusion conditions by using 0.8 M KH2PO4, 0.8 M molybdenum-containing iron-sulfur flavoprotein that cata- NaH2PO4, 2% MPD, and 100 mM Hepes adjusted to pH 7.3. ϩ 3 ϩ Ϫ ϩ ϩ lyzes the oxidation of CO (CO H2O CO2 2e 2H ) Crystals containing cyanide were cocrystallized in the pres- and generates a proton gradient across the cytoplasmic mem- ence of 4 mM potassium cyanide. For synchrotron data brane by channeling the electrons formed via cytochrome b561 collection under low-temperature conditions, 15% glycerol into a CO-insensitive respiratory chain (4). O. carboxidovorans was added to the crystallization buffer. Before measurements, uses the reactions of the Calvin cycle for CO2 fixation (4). In the concentration of glycerol was gradually increased to 25%. the exponential growth phase, cells of O. carboxidovorans have The crystals had an orthorhombic space group P212121 with 87% of CO dehydrogenase associated with the inner aspect of cell dimensions of a ϭ 129.14 Å, b ϭ 140.34 Å, and c ϭ 164.44 the cytoplasmic membrane and 13% located in the cyto- Å and two monomers per asymmetric unit. plasm (6). Structure Determination. With a first data set to 3.0 Å, a CO dehydrogenase is composed of the 88.7-kDa L (809 Patterson search was carried out. The program AMORE (13) residues), 30.2-kDa M (288 residues), and 17.8-kDa S (166 gave a clear solution for a search model constructed from residues) subunits and exists as a dimer of LMS heterotrimers homologous parts of Mop, which comprised a total of about (4). The L subunit carries the molybdenum cofactor, which is 10% of the scattering mass of the asymmetric unit [R factor of a mononuclear complex of Mo and molybdopterin–cytosine 50.8%, with a correlation coefficient (cc) of 29.4 after trans- dinucleotide (MCD) (7). FAD-binding is on the M subunit and Abbreviations: MCD, molybdopterin–cytosine dinucleotide; VAO, The publication costs of this article were defrayed in part by page charge vanillyl–alcohol oxidase. Data deposition: The atomic coordinates have been deposited in the payment. This article must therefore be hereby marked ‘‘advertisement’’ in Protein Data Bank, www.rcsb.org (PDB ID code 1QJ2). accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed. E-mail: dobbek@ PNAS is available online at www.pnas.org. biochem.mpg.de.

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lational search for the second monomer, compared with the next highest peak with an R factor of 52.1% and a cc of 24.7 in a resolution range of 14.0–5.0 Å]. Because it was not possible to interpret the calculated density, we initiated multiwavelength anomalous diffraction data collection at two wavelengths (remote wavelength and iron K-edge wavelength) by using synchrotron radiation at BW6, DESY, Hamburg. Data sets native1 and native2 were collected on a MAR-Research (Hamburg, Germany) charge-coupled device detector at Ϫ165°C at BW6, DESY and processed by using DENZO and SCALEPACK (14). By using an anomalous difference Fourier map calculated with the phases obtained from the Patterson search solution, the positioning of the four [2Fe–2S] clusters could be achieved. The phasing was carried out by using MLPHARE (15) and SHARP (16) and allowed after solvent flattening and histogram matching the recognition of the molecular boundaries. A phase combination with subsequent solvent flattening and cyclic averaging within the dimer was performed and allowed us to calculate an interpretable elec- FIG. 1. Ribbon representation of the CO dehydrogenase dimer. tron-density map from the two phase sources. For phase The twofold-symmetry axis of the dimer runs vertically in the plane of ␤ modification, the CCP4 (15) package was used. Density inter- the figure. Helices are shown in red, -sheets in blue, and the connecting turns and loops in yellow. The cofactors are depicted in pretation and model building were carried out by using MAIN white. Structure presentations were created by using BOBSCRIPT and (17). The structure was refined by using torsion-angle dynam- RASTER3D. ics, simulated annealing, and maximum likelihood targets for positional refinement as provided by CNS (18). occurs in a redox state that is reduced by two electrons The cyanide-cocrystallized crystal was measured on a compared with the fully oxidized state (19), a tricyclic tetra- MAR-Research (Hamburg, Germany) 300 image plate detec- hydropterin–pyran system. The MCD–molybdenum cofactor ␣ tor at room temperature by using monochromatized CuK- is located in the L subunit, and the two [2Fe–2S] clusters are radiation produced by a conventional rotating anode (Table 1). located in the S subunit (Fig. 2). These prosthetic groups form a pathway for the electrons to the FAD located in the M RESULTS subunit (Fig. 2). The two active sites of the dimer are at a Mo–Mo distance of 52.9 Å with no further cofactors located Overall Structure. CO dehydrogenase is dimeric in solution, between them, indicating two separate catalytic units in CO and two monomers were found in the asymmetric unit. The dehydrogenase. butterfly-shaped dimer (Fig. 1) has overall dimensions of Structure of the Three Subunits. Subunit S represents the ϫ ϫ 2 145 100 70 Å with an accessible surface area of 78,076 Å . iron-sulfur protein of CO dehydrogenase and is clearly divided The two monomers are basically identical, with an rms devi- ␣ into a C- and an N-terminal domain, each binding a [2Fe–2S] ation of 0.30 Å for the C atoms. cluster, which can be distinguished by EPR (8). The C-terminal The redox components of one LMS-structured monomer domain (residues 77–161) carries the proximal [2Fe–2S] clus- are the MCD–molybdenum cofactor, composed of a molyb- ter. The cluster is buried in CO dehydrogenase Ϸ11 Å below denum ion with two oxo- and one hydroxoligand, complexed the protein surface at the interface between the S and the L by the enedithiolene group of MCD, [2Fe–2S] clusters of type subunit and is adjacent to the MCD–molybdenum cofactor. I and type II, and a noncovalently bound FAD molecule (Fig. The [2Fe–2S] cluster is located at the N terminus of two 2 C and D). In air-oxidized CO dehydrogenase, the flavin is ␣ ϩ -helices that participate in a four-helix bundle of twofold fully oxidized, the oxidation state of Mo is VI, and MCD symmetry, an architecture first described for the Mop protein (11). Table 1. Data collection and refinement statistics The N-terminal domain (residues 3–76) is similar to those of Native 1͞2 Cyanide plant-type [2Fe–2S]-ferredoxins (20). It carries the distal Data collection [2Fe–2S] cluster, which is adjacent to the FAD residing on M. Wavelegth, Å 0.9761͞1.7316 1.5418 Although the cluster is positioned at the interface between S Resolution limit, Å 2.2͞2.5 2.36 and M, it is exposed to the solvent and mediates electron Unique reflections 144,247͞97,346 105,076 transfer from the proximal [2Fe–2S] cluster to the isoallox- Redundancy 3.4͞3.8 4.1 azine ring of FAD. Completeness, % 92.6͞89.2 92.5 Subunit M is the flavoprotein of CO dehydrogenase. It binds Rsymm* 0.034͞0.053 0.119 FAD in a 1:1 molar stoichiometry (Fig. 5 A and B) and exhibits Refinement the motifs 32MAGGHS36M and 111MTIGG114M typical of the Protein non-hydrogen atoms 19,098 19,102 FAD-binding site of the vanillyl-alcohol oxidase family of Solvent molecules 1,407 783 flavoproteins. Both motifs are conserved in the molybdenum Resolution range, Å 25.0–2.2 38.0–2.36 hydroxylase sequence family (Fig. 5C). M is arranged in three Total reflections, F Ͼ 2␴F 14,1156 10,4855 distinct domains (Fig. 5 B and C), designated the N-terminal Total reflections used in Rfree 4,251 3,169 domain (residues 1–54), the middle domain (residues 60–174), Rcryst,† % 18.8 17.0 and the C-terminal domain (residues 180–285). The N- Rfrec,‡ % 23.1 20.5 terminal domain is composed of a three-stranded parallel rms deviation of bond distance, Å 0.0086 0.0059 ␤-sheet (␤1–␤3) flanked by two helices (␣1–␣2). A loop rms deviation of angles, ° 1.68 1.56 between ␣2 and ␤2ofa␤␣␤-unit (P loop, residues 32–36) contains one of the two double-glycine motifs, which interact *Rsymm ϭ͚͉I Ϫ͗I͉͚͘͞I. †Rcryst ϭ͚ʈFabs͉ Ϫ ͉Fcalcʈ͚͉͞Fabs͉. with the pyrophosphate moiety of the FAD molecule. The ‡Rfree is the same as Rcryst calculated with a test set comprising 5% of main structural element of the middle domain is a five- the whole data set that was not used in the refinement. stranded antiparallel ␤-sheet (␤4–␤8) that contains an inser- Downloaded by guest on September 27, 2021 8886 Biochemistry: Dobbek et al. Proc. Natl. Acad. Sci. USA 96 (1999)

FIG. 2. Comparison of the CO dehydrogenase monomer (B) with the monomeric Mop structure from Desulfovibrio gigas (12) (A). Domain structure (C) and cofactors (D) of the CO dehydrogenase monomer. The ribbon presentation shows the general similarity of the two monomers for the MCD–molybdenum cofactor-containing subunit/domain (white) and the iron-sulfur containing subunit/domain (red) in both enzymes. The FAD-containing M subunit (yellow) is absent in the Mop protein. Whereas the S subunits are very similar in both proteins, the L subunit differs in a few areas. This applies mainly to the substrate channel of Mop (white arrow), which is closed by an additional loop in CO dehydrogenase and to the first part of the C-terminal domain (yellow encircled), which creates the dimer interface. (C) The three subunits of CO dehydrogenase may be subdivided into domains (the L subunit in a cyan N-terminal domain and a blue C-terminal domain). The S subunit has the iron-sulfur clusters bond in the N-terminal domain drawn in red and in the C-terminal domain drawn in orange. The three domains of the M subunit are yellow, white, and cream, sequentially, from N to C terminus. (D) The shortest connection between the cofactors is marked by a white line, starting from the molybdo-oxo group (MCD–Mo) at the active site via the proximal C-terminal [2Fe–2S] cluster (FeS I) (14.6 Å distance from the Mo atom to the closest iron atom) and the distal N-terminal [2Fe–2S] cluster (FeS II) (12.4 Å distance between the closest iron atoms), ending at the FAD (8.7 Å distance between C7 of FAD and the closest iron atom).

tion of six small ␣-helices winding around the adenosine respect to the architecture of structural elements interacting dinucleotide portion of the FAD molecule. A small ␣-helix with FAD. MurB (UDP–N-acetylenolpyruvylglucosamine re- contains the second double-glycine motif (residues 111–115), ductase) of Escherichia coli (22) showed a Z score of 8.9 by which also interacts with the FAD. The N-terminal and the using the fold of M as a search model. The vanillyl–alcohol middle domains form the wings holding the cofactor (Fig. 5B). oxidase (VAO) of Penicillium simplicissimum (23) gave a Z The C-terminal domain contains a three-stranded antipar- score of 6.0. VAO is a member of a new oxidoreductase family allel ␤-sheet (␤10–␤12) that merges in a bundle of three sharing a conserved FAD-binding domain (24). The fold of ␣-helices (␣8–␣10). It is obvious that the isoalloxazine ring is MurB and VAO involved in binding the dinucleotide moiety of accessible only from one side of the M subunit near the subunit the FAD cofactor resembles the N-terminal and middle do- interface. The central part of the isoalloxazine ring is shielded main of the M subunit of CO dehydrogenase. The residues from the solvent by the side chain of Tyr193M, which is part of forming two loops responsible for binding the FAD in the an extended loop created between ␤10 and ␤11 (Q loop, CoxM polypeptide are mainly conserved in the molybdenum residues 192–195) of the C-terminal domain (Fig. 5 B and C). hydroxylase enzyme family (Fig. 5C) and can also be found as The distal parts of the dimethyl benzene ring remain solvent conserved fingerprints in the new VAO family. Both loops accessible. This applies to the C7 and C8 positions with their have in their central portion two small amino acids, often a methyl groups as well as for the C9 position of the isoalloxazine double glycine. The first binding motif lying on the P loop can ring. The C4a and N5 atoms may become accessible by be defined as aGgt, with the sequence 32AGGH35 in CoxM. movements of the C-terminal domain or the side chain of The second motif on ␣6 and ␣7 can be defined as AxpqiRnx- Tyr193M. The C-terminal domain is linked to the middle atxGGn, equivalent to 102ADPQIRYMGTIGGN115 in CoxM domain by a flexible loop that could provide the flexibility. (Fig. 5C). In the VAO family, a ssGHs and a shsG motif are The C-terminal domain interacts only with the isoalloxazine responsible for binding the pyrophosphate moiety of FAD ring at its O4 position. (24). It is likely that other flavin-containing molybdenum By using the program DALI (21), two proteins were identified hydroxylases exhibit flavin-binding sites similar to that ob- with similarity to the M subunit of CO dehydrogenase with served in CO dehydrogenase. Downloaded by guest on September 27, 2021 Biochemistry: Dobbek et al. Proc. Natl. Acad. Sci. USA 96 (1999) 8887

FIG. 3. The molybdenum site of the L subunit. (A) The Mo ion exhibits one hydroxo and two oxo ligands and is bound to MCD through its enedithiolene group. The second coordination sphere of the Mo ion is composed of the residues Gln240L, Glu763L, and S-selanyl-Cys388L. B and C show the active site together with the adjacent [2Fe–2S] cluster. Difference Fourier maps were calculated with the phases from the final model shifted by 90° (imaginary map) and with amplitudes from the Bijvoet differences at a wavelength ␭1 ϭ 1,7316 Å (B) shown in red and calculated with data obtained at a wavelength of ␭2 ϭ 0,9761 Å (C) shown in yellow. The maps are contoured at 3␴. Calculated fЈЈ values using the program ABSORB for selected Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ Ϫ free atoms are Fe: ␭1, 3.9 e and ␭2, 1.5 e ; Se: ␭1, 1.41 e and ␭2, 3.84 e ;S:␭1, 0.69 e and ␭2, 0.23 e ; Mo: ␭1, 3.36 e and ␭2, 1.23 e .

The C-terminal domains of the CO dehydrogenase M coordination sphere contains a modified cysteine residue subunit and its counterparts in MurB and VAO are structurally (Cys388L, Fig. 3 A–C). Strong additional density between unrelated and may convey class-specific properties. Cys388L and the equatorial oxo group was modeled as a SeH Subunit L has a heart-like shape with dimensions of 60 ϫ group attached to the S␥ of the cysteine residue as an 83 ϫ 70 Å and contains two subdomains (Fig. 2C). Its S-selanylcysteine. The identification of the selenium is based ␭ N-terminal domain (residues 10–439) interacts with the M and on the anomalous scattering at two different wavelengths ( 1 ϭ ␭ ϭ S subunits in the same monomer. Its fold is dominated by a 0.976 Å and 2 1.732 Å, Fig. 3 B and C) and agrees with five- and a four-stranded mixed ␤-sheet. The C-terminal data of chemical analyses (ref. 25; unpublished results). The domain (residues 440–809) can be further subdivided into two selenium atom of S-selanylcysteine is located in a distance of parts. The first part consists of a central ␣-helix of 30 aa that 3.7 Å from the Mo ion. It is near the equatorial oxo and runs into a small three-stranded ␤-sheet followed by an ␣-helix hydroxo group of the Mo ion. The modified residue is situated and a four-stranded parallel–antiparallel ␤-sheet, which inter- at a loop that shows an amino acid composition unique for the acts with its symmetrymate of the other monomer, providing CO dehydrogenases, because its sequence of VAYRC388LSFR the main dimer contact. The second part of the C-terminal is also present in the sequence of CO dehydrogenase from domain consists of a long three-stranded antiparallel ␤-sheet Pseudomonas thermocarboxydovorans or Hydrogenophaga surrounded by seven ␣-helices. pseudoflava but absent in other enzymes of the molybdenum Active Site with S-Selanylcysteine. The active site is acces- hydroxylase family. The active-site loop occupies the position sible from the outside through a narrow channel that is 17 Å of the substrate-analog isopropanol bound in the Mop struc- deep with an average diameter of 7 Å, determined from the C␤ of the surrounding residues. A restriction of the active-site channel may exclude substrates larger than CO and may prevent dissociation of the cofactor from the protein. The character of the channel is hydrophobic, so that the first charged residues an incoming molecule can interact with are the protein ligands around the molybdenum in its second coordination sphere. The MCD–molybdenum cofactor is buried at the center of the L subunit (Fig. 2C) and is ligated through a dense network of hydrogen bonds originating from both domains of L. The geometry of the first coordination sphere around the Mo ion can be described as a distorted square pyramid (Fig. 3A). The dithiolene group of MCD is positioned in the equatorial plane together with two oxygen atoms, which were modeled as an oxo- and a hydroxo- group. The apical ligand was modeled as an oxo- group. In contrast to xanthine oxidases/dehydrogenases, active CO dehydrogenase does not contain a cyanolys- able sulfido ligand at the Mo ion. The apical oxo-group can form a hydrogen-bond to the N␧2 of Gln240L, located in a distance of 3.0 Å. Gln240L is highly conserved in the molybdenum hydroxylase family, although it

has no equivalent in the Mop protein. FIG. 4. Hypothetical reaction mechanism. Product CO2 may re- Trans to the apical oxo group is a conserved glutamate main in the metal coordination sphere such that the number of oxygen residue (Glu763L) with a Mo–O␧1 distance of 3.1 Å. The second ligands is unchanged during the catalytic steps. Downloaded by guest on September 27, 2021 8888 Biochemistry: Dobbek et al. Proc. Natl. Acad. Sci. USA 96 (1999)

FIG. 5. Structure of the M subunit. (A) Molecular interactions of FAD in the M subunit. Atoms of the isoalloxazine ring are labeled. Residues binding the FAD cofactor through hydrogen bonds are marked, and boxes around the labels give the colors of the domains that harbor the corresponding residue. Whereas the N-terminal domain (yellow boxes) binds only to the dinucleotide portion of FAD, the middle domain (red boxes) interacts also with the riboflavin part. The C-terminal domain (blue box) shows only a single hydrogen bond to FAD. (B) Fold of the M subunit. The N-terminal domain is shown in yellow, the middle domain in red, and the C-terminal domain in blue. Tyr193M shields the isoalloxazine ring and is shown in cyan. The positions of residues corresponding to the rosy mutant strains of Drosophila melanogaster that resulted in an altered affinity to bind NADϩ in xanthine dehydrogenase are shown as green enlargements of the C␣ chain and have a green circle under their position in C.(C) Sequence alignment of the M polypeptide of CO dehydrogenase with the corresponding representative parts from other members of the molybdenum hydroxylase family: CODH, CO dehydrogenase from Oligotropha carboxidovorans (coxM, Swall: Q51323); QuinOR, quinoline 2-oxidoreductase from Pseudomonas putida (qorM, Swall: P72222); NicotDH, nicotine dehydrogenase from Arthrobacter nicotinovorans (ndhA, Swall: Q59127); XDHRC, xanthine dehydrogenase from Rhodobacter capsulatus (xdhA, Swall: O54050); XDHBM, xanthine dehydrogenase from Bombyx mori (Swall: Q17209); XDHCV, xanthine dehydrogenase from Calliphora vicina (xdh, Swall: XDH CALVI); XDHDM, xanthine dehydrogenase from Drosophila melanogaster (xdh, Swall: XDH DROME). Two boxes highlighting residues responsible for FAD binding in the M subunit are shown in red and blue. They correspond to fingerprints for FAD-binding by enzymes of the molybdenum hydroxylase family and define a FAD-binding motif similar to the motifs found for the vanillyl alcohol oxidase family of flavoproteins. The secondary structure is represented by arrows for ␤-sheets and bars for ␣-helices, in domain colors (B). Residues appearing at least three times in a row are colored in green. The position of Tyr193M is marked by a cyan circle below the aligned sequences.

ture and could be involved in substrate-binding in CO dehy- anoxic conditions (half-life of 1 h, complete inactivation after drogenase. 10 h). Enzyme crystallized in the presence of 4 mM potassium cyanide showed a reduced occupancy, with selenium at the S-selanylcysteine residue in the structure, and the remaining DISCUSSION selenium now appears in a small linear molecule interpreted as Hypothetical Reaction Mechanism. The chemistry of carbon selenocyanate. The molecule lies in the equatorial plane of the oxide selenide (OACASe) and the selenium-catalyzed syn- oxygen ligands, and the selenium is in hydrogen-bond distance 388L 385L thesis of carbonates from CO (26) show remarkable analogies to the amide nitrogen atoms of Cys and Ala . Because CO and cyanide are isoelectronic, isosteric, and show a similar to the CO dehydrogenase-catalyzed oxidation of CO to CO2. SeCO is a gas, which is easily prepared through the oxidation chemical reactivity, we assume a similar reaction between CO of CO with elemental selenium in the presence of a strong base and selenium, leading to a carbonyl selenide that reacts with at normal pressure and room temperature (26). When SeCO water to yield CO2 (Fig. 4). The catalytic mechanism of CO dehydrogenase thus can be is treated with a sodium alcoholate (RONa) under mild imagined to involve the formation of carbon oxide selenide conditions, the corresponding sodium O-alkyl selenocarbonate Ϫ ϩ with the subsequent nucleophilic attack by the Mo–OH group, (ROCAOSe Na ) is formed, which undergoes nucleophilic yielding CO2 (Fig. 4). Catalysis in CO dehydrogenase starts by attack by the alcohol, yielding the corresponding dialkyl 388L A ϭ the reaction of CO with the selenium of S-selanylcysteine , carbonate [(RO)2C O]. In aqueous sodium hydroxide (r thereby forming a selenocarbonyl species (Fig. 4). The seleno- H), carbon oxide selenide reacts to carbonate and selenide carbonyl undergoes a nucleophilic attack by the Mo–OH group ϩ 3 ϩ ϩ (COSe 4 NaOH Na2Se Na2CO3 2H2O). of the molybdenum, which is in the oxidized ϩVI state. The active site of CO dehydrogenase literally assembles all Subsequently, two electrons are abstracted, and CO2 is re- components essential in the selenium-catalyzed synthesis of leased. Electron transfer from the reduced Mo ion in the ϩIV carbonates (Fig. 3). The selenium of the S-selanylcysteine388L state to the proximal [2Fe–2S] center and the addition of water is formally elemental Se with the redox state 0 (Fig. 4). It reacts regenerates the oxidized Mo–OH in the ϩVI oxidation state. with cyanide, yielding selenocyanate, with the concomitant The transport of the electrons from the active-site Mo ion to irreversible inactivation of CO dehydrogenase under oxic or the external electron acceptors is facilitated through a chain of Downloaded by guest on September 27, 2021 Biochemistry: Dobbek et al. Proc. Natl. Acad. Sci. USA 96 (1999) 8889

redox active cofactors composed of two [2Fe–2S] clusters and Grant Me 732/5-3 from the Deutsche Forschungsgemeinschaft (Bonn- FAD. The MCD–molybdenum cofactor is adjacent to the Bad Godesberg, Germany) to O.M. proximal [2Fe–2S] cluster located in the C-terminal domain of the S subunit (Fig. 2D). The molybdopterin ring is positioned 1. Sanhueza, E., Dong, Y., Scharffe, D., Lobert, J. M. & Crutzen, between the molybdenum and the [2Fe–2S] cluster and sup- P. J. (1998) Tellus, Ser. B 50, 51–58. 2. Moxley, J. M. & Smith, K. A. (1998) Soil Biol. Biochem. 30, 65–79. ports electron transfer through its conjugated double bonds. Ј 3. Mo¨rsdorf, G., Frunzke, K., Gadkari, D. & Meyer, O. (1992) The N2 amino group of the fused pyrimidin ring is at a Biodegradation 3, 61–82. distance of 5.5 Å from the Fe1 atom of the proximal cluster. 4. Meyer, O., Frunzke, K. & Mo¨rsdorf, G. (1993) Microbial Growth CO Dehydrogenase as a Member of the Xanthine Oxidase on C1 Compounds, eds. Murrell, J. C. and Kelly, D. P. (Intercept, Family. The CO dehydrogenase reveals significant sequence Andover, U.K.), pp. 433–459. homologies with the other members of the molybdenum 5. Ragsdale, S. W. & Kumar, M. (1996) Chem. Rev. 96, 2515–2539. hydroxylase family, suggesting structural conservation and 6. Rohde, M. Mayer, F. Meyer, O. (1984) J. Biol. Chem. 259, 14788–14792. represents, therefore, a close structural model of the eukary- 7. Meyer, O., Frunzke, K., Tachil, J. and Volk, M. (1993) in otic xanthine oxidase/dehydrogenase in tertiary and quarter- Molybdenum Enzymes, Cofactors, and Model Systems, eds. Stiefel, nary structure. Mop had served as a first approximation to the E. I., Coucouvanis, D. & Newton, D. W. E. (Am. Chem. Soc., structure of the xanthine oxidase/dehydrogenase (11), but Washington, DC), pp. 433–459. lacked the flavin domain that is now defined in CO dehydro- 8. Bray, R. C. George, G. N. Lange, R. & Meyer, O. (1983) genase by the M subunit. Its location differs from the position Biochem. J. 211, 687–694. of the connecting loop in Mop suggested to be a marker (11). 9. Hille, R. (1996) Chem. Rev. 96, 2757–2816. Major gaps in sequence alignments between the enzymes are 10. Schu¨bel, U., Kraut, M. & Mo¨rsdorf, G. (1995) J. Bacteriol. 177, 2197–2203. due to the fact that most of the xanthine oxidases/ 11. Romao, M. J., Archer, M., Moura, I., Mowra, J. J. G., LeGall, J., dehydrogenases, as well as Mop, contain connecting segments Engh, R., Schneider, M., Hof, P. & Huber, R. (1995) Science 270, between their structural domains, which are missing in the 1170–1176. heterotrimeric CO dehydrogenase. 12. Santiago, B. & Meyer, O. (1997) J. Bacteriol. 179, 6053–6060. The homology between CO dehydrogenase and other mem- 13. Navazza, J. (1994) Acta Crystallogr. A 50, 157–163. bers of the molybdenum hydroxylase family extends beyond to 14. Otwinowski, Z. (1993) in Data Collection and Processing, eds. residues in the core of the domains/subunits to the interdo- Sawyer, L., Isaacs, N. & Bailey, S. (Science and Engineering main/subunit contacts. In addition, the arrangement of the L Research Council, Daresbury Laboratory, Warrington, U.K.), pp. 56–62. and the S subunit of CO dehydrogenase is identical to the 15. Collaborative Computational Project No. 4 (1994) Acta Crystal- arrangement of the corresponding parts of Mop (Fig. 2). logr. D 50, 760–763. The major functional difference between the interconvert- 16. de la Fortelle, E. & Bricogne, G. (1997) Methods Enzymol. 276, ible forms xanthine oxidase and xanthine dehydrogenase is 472–493. their usage of electron acceptors. These different reactivities 17. Turk, D. (1992) Ph.D. thesis (Technische Universita¨t Mu¨nchen, may be correlated to the ability of binding NADϩ/NADH. Germany). Affinity-labeling studies with the pyridine nucleotide analog 18. Bruenger, A. T. Adams, P. D., Clore, G. M., DeLano, W. L., 5Ј-p-fluorosulfonylbenzoyladenosine (5Ј-FSBA) (27) hinted to Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., ϩ Nilges, M., Pannu, N. S., et al. (1998) Acta Crystallogr. D 54, a tyrosine residue affecting NAD binding. Reactivity studies 905–921. of xanthine dehydrogenase variants from rosy mutant strains of 19. Tachil, J. & Meyer, O. (1997) FEMS Microbiol. Lett. 148, Drosophila melanogaster comprised mutants that showed a 203–208. diminished affinity for NADϩ or were unable to use NADϩ as 20. Sticht, H. & Roesch, P. (1998) Prog. Biophys. Mol. Biol. 70, an electron acceptor (28). Through sequence comparisons 95–136. between the xanthine oxidase/dehydrogenase sequences from 21. Holm, L. & Sander, C. (1993) J. Mol. Biol. 233, 123–138. different sources with the sequence of the CO dehydrogenase 22. Benson, T. E., Filman, D. J., Walsh, C. T. & Hogle, J. M. (1995) Nat. Struct. Biol. 2, 644–653. M subunit, we localized in our model the positions of these 23. Mattevi, A., Fraaije, M. W., Mozzarelli, A., Olivi, L., Coda, A. & mutations and the labeled tyrosine residue. They correspond van Bokel, W. J. H. (1997) Structure (London) 5, 907–920. 119M 123M 156M to Gly , Asn , and Ala in CoxM (Fig. 5 B and C). All 24. Fraaije, M. W., Berkel, W. J. H., van Benen, J. A. E., Visser, J. of these residues cluster around two loops of the M subunit & Mattevi, A. (1998) Trends Biochem. Sci. 23, 206–207. near the solvent-exposed side of the isoalloxazine ring of the 25. Meyer, O. & Rajagopalan, K. V. (1984) J. Biol. Chem. 259, FAD. Their mutation may alter the ability to bind NADϩ. The 5612–5617. likely docking site of NADϩ suggests hydride transfer from the 26. Paulmier, C. (1986) Selenium Reagents and Intermediation in isoalloxazine ring to a pyridine ring positioned at the si face. Organic Synthesis (Pergamon, Oxford). 27. Nishino, T. & Nishino, T. (1987) Biochemistry 26, 3068–3072. This unusual stereochemistry was also suggested for the in- ϩ 28. Doyle, W. A., Burke, J. F., Chovnick, A., Dutton, F. L., Whittle, teraction of MurB with NADP (29). J. R. S. & Bray, R. C. (1996) Eur. J. Biochem. 239, 782–795. 29. Constantine, K. L., Mueller, L., Goldfarb, V., Wittekind, M., We thank Dr. H. Bartunik and G. B. Bourenkow at the BW6, DESY, Metzler, W. J., Yanchunas J., Jr., Robertson, J. G., Malley, M. F., Hamburg for their help in data collection and multiwavelength Fridericks, M. S. & Farmer, B. T., II. (1997) J. Mol. Biol. 267, anomalous diffraction measurements. This work was supported by 1223–1246. Downloaded by guest on September 27, 2021