CO dehydrogenase (EC 1.2.99.2) catalyzes the oxidation of CO according to the following equation: CO + H2O-->CO2 + 2 e- + 2 H+. It is a selenium-containing molybdo-iron-sulfur-flavoenzyme, which has been crystallized and structurally characterized in its oxidized state from the aerobic CO utilizing bacteria Oligotropha carboxidovorans and Hydrogenophaga pseudoflava. Both CO dehydrogenase structures show only minor differences, and the enzymes are dimers of two heterotrimers. Each heterotrimer is composed of a molybdoprotein, a flavoprotein, and an iron-sulfur protein. CO oxidation takes place at the molybdoprotein which contains a 1:1 mononuclear complex of molybdopterin-cytosine dinucleotide and a Mo-ion, along with a catalytically essential S-selanylcysteine. The latter is appropriately positioned in the SeMo-active site by a unique VAYRCSFR active site loop. In H. pseudoflava the arginine preceeding the cysteine in the active site loop is modified to a Cgamma-hydroxy arginine residue which has no obvious function. The substituents in the first coordination sphere of the Mo-ion are the enedithiolate sulfur atoms of the molybdopterin-cytosine dinucleotide, two oxo- and a sulfido-group. Extended X-ray absorption fine structure spectroscopy (EXAFS), along with the crystal structure of CO dehydrogenase (23.2 U mg(-1)) at 1.85 A resolution, have identified a sulfur atom at 2.3 A from the Mo-ion. The sulfur reacts with cyanide yielding thiocyanate. The corresponding inactive desulfo-CO dehydrogenase shows a typical desulfo inhibited-type of Mo-electron paramagnetic resonance (EPR) spectrum. Structural changes at the SeMo-site during catalysis are suggested by the Mo to Se distance of 3.7 A and the Mo-S-Se angle of 113 degrees in the oxidized enzyme which increase to 4.1 A, and 121 degrees, respectively, in the reduced enzyme. The intramolecular electron transport chain in CO dehydrogenase involves the following prosthetic groups and minimal distances: CO-->[Mo of the molybdenum cofactor] - 14.6 A - [2Fe-2S]I - 12.4 A - [2Fe-2S]II - 8.7 A - [FAD].
CO dehydrogenase from the Gram-negative chemolithoautotrophic eubacterium Oligotropha carboxidovorans OM5 is a structurally characterized molybdenum-containing iron-sulfur flavoenzyme, which catalyzes the oxidation of CO (CO ؉ Oligotropha carboxidovorans OM5 is an aerobic, Gramnegative member of the ␣-subclass of proteobacteria (1, 2). It utilizes CO as a sole source of carbon and energy under chemolithoautotrophic conditions (carboxidotrophy). The 133-kbp circular DNA megaplasmid pHCG3 of O. carboxidovorans has been completely sequenced (3), and the annotated genome sequence of the chromosome has been reported (4). The plasmid carries the gene clusters cox, cbb, and hox, which assemble the functions required for the utilization of CO, CO 2 , or H 2 , respectively (3). The three clusters form a 51.2-kb chemolithoautotrophy module. Transcription of the cox gene cluster (Fig. 1A) requires the presence of CO (3,5,6). CO dehydrogenase is the key enzyme in the utilization of CO {CO ϩ H 2 O 3 CO 2 ϩ 2e Ϫ ϩ 2H ϩ } (7). It is a molybdenum-and copper-containing ironsulfur flavoenzyme. The subunit structure is a dimer of heterotrimers, which are encoded by the genes coxMSL (3, 5, 6). The L subunit (CoxL, 88.7 kDa) is a molybdo-copper protein.HIt accommodates the catalytic site, which is buried ϳ17 Å below the solvent-accessible surface of CO dehydrogenase (8 -10). CO is oxidized at a unique [CuSMoO 2 ] cluster.The molybdenum ion is coordinated by the molybdopterin cytosine dinucleotide cofactor (7,8,11). The copper ion is coordinated by the cysteine residue 388 of the active site loop VAY-RCSFR (5, 6).The M-subunit (CoxM, 30.2 kDa) is a flavoprotein accommodating the flavin adenine dinucleotide (FAD) 4 cofactor. Binding of FAD to the flavoprotein requires the heterotrimeric enzyme complex (8, 12). The S-subunit (CoxS, 17.8 kDa) is an iron-sulfur protein that contains one [2Fe-2S] center proximal to the [CuSMoO 2 ] cluster and another distal one. These cofactors establish an intramolecular electron transport that delivers the electrons generated through the oxidation of CO at the [CuSMoO 2 ] cluster to [2Fe-2S] I, [2Fe-2S] II, and finally to FAD, from where they are fed into a CO-insensitive respiratory chain to generate a membrane potential (13).O. carboxidovorans synthesizes CO dehydrogenase in two forms: as the catalytically active enzyme species and as a properly assembled apo-enzyme, which does not oxidize CO (10). Usually, both mature and immature species of the enzyme co-exist in the bacterial cell. Other than the functional enzyme, which contains the [CuSMoO 2 ] cluster, the apo-enzyme lacks the copper ion and/or the sulfur connecting the two metals (10,14
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