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Information on EC 1.2.5.3 - aerobic carbon monoxide dehydrogenase and Organism(s) Afipia carboxidovorans and UniProt Accession P19921
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1.2.5.3 aerobic carbon monoxide dehydrogenaseIUBMB Comments
This enzyme, found in carboxydotrophic bacteria, catalyses the oxidation of CO to CO2 under aerobic conditions. The enzyme contains a binuclear Mo-Cu cluster in which the copper is ligated to a molybdopterin center via a sulfur bridge. The enzyme also contains two [2Fe-2S] clusters and FAD, and belongs to the xanthine oxidoreductase family. The CO2 that is produced is assimilated by the Calvin-Benson-Basham cycle, while the electrons are transferred to a quinone via the FAD site, and continue through the electron transfer chain to a dioxygen terminal acceptor . cf. EC 1.2.7.4, anaerobic carbon monoxide dehydrogenase.
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The taxonomic range for the selected organisms is: Afipia carboxidovorans
The enzyme appears in selected viruses and cellular organisms
The enzyme appears in selected viruses and cellular organisms
Synonyms
mocu-codh, aerobic carbon monoxide dehydrogenase, mo-cu carbon monoxide dehydrogenase, molybdenum-containing co dehydrogenase, molybdoenzyme carbon monoxide dehydrogenase, molybdenum-containing carbon monoxide dehydrogenase, molybdenum- and copper-dependent co dehydrogenase, 
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topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
CO dehydrogenase
CODH
coxSML
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MoCu-CODH
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molybdenum- and copper-dependent CO dehydrogenase
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molybdenum-containing carbon monoxide dehydrogenase
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molybdenum/copper-containing carbon monoxide dehydrogenase
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molybdoenzyme carbon monoxide dehydrogenase
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REACTION
REACTION DIAGRAM
COMMENTARY
ORGANISM
UNIPROT
LITERATURE
CO + a quinone + H2O = CO2 + a quinol
CO initially binds rapidly to the enzyme, possibly at the Cu(I) of the active site, prior to undergoing oxidation. A Mo(V) species exhibits strong coupling to the copper of the active center, the rate-limiting step of overall turnover is likely in the reductive half-reaction
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CO + a quinone + H2O = CO2 + a quinol
hypothetical reaction mechanism, overview
CO + a quinone + H2O = CO2 + a quinol
proposed reaction mechanisms for CO dehydrogenase, the rate-limiting step for overall turnover resides in the reductive half-reaction, reoxidation of reduced enzyme by quinones occurs at the FAD site
CO + a quinone + H2O = CO2 + a quinol
reaction mechanism that initially involves nucleophilic attack of a Mo=O oxo on the carbon center of Cu(I)-CO, resulting in a 5-membered cyclic m2-e(2) CO2-bridged Mo(VI)-Cu(I)-Cys complex I that can bind HO-/H2O to yield 1-OH. This is followed by a second nucleophilic attack on the activated mu2-nu2 CO2 carbon centre of 1-OH to yield a Mo(IV)-bicarbonate product complex, 1-P. This second nucleophilic attack is suggested based on our electronic structure description of cyclic m2-e(2) CO2-bridged Mo(VI)-Cu(I)-Cys complex I, which possesses a bent and activated CO2 bound to the Mo and Cu ions. Proposed catalytic cycle for CODH that avoids formation of a stable C-S bonded cyclic m2-e(2) CO2-bridged Mo(VI)-Cu(I)-Cys complex II
PATHWAY SOURCE
PATHWAYS
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SYSTEMATIC NAME
IUBMB Comments
carbon-monoxide:quinone oxidoreductase
This enzyme, found in carboxydotrophic bacteria, catalyses the oxidation of CO to CO2 under aerobic conditions. The enzyme contains a binuclear Mo-Cu cluster in which the copper is ligated to a molybdopterin center via a sulfur bridge. The enzyme also contains two [2Fe-2S] clusters and FAD, and belongs to the xanthine oxidoreductase family. The CO2 that is produced is assimilated by the Calvin-Benson-Basham cycle, while the electrons are transferred to a quinone via the FAD site, and continue through the electron transfer chain to a dioxygen terminal acceptor [5]. cf. EC 1.2.7.4, anaerobic carbon monoxide dehydrogenase.
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
CO + 1,2-naphthoquinone-4-sulfonic acid + H2O
CO2 + 1,2-naphthoquinol-4-sulfonic acid
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-
-
?
CO + 1,4-naphthoquinone + H2O
CO2 + 1,4-naphthoquinol
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-
-
?
CO + 2-(4-iodophenyl)-3-(4-nitrophenyl)-2H-tetrazolium chloride + H2O
CO2 + reduced 2-(4-iodophenyl)-3-(4-nitrophenyl)-2H-tetrazolium chloride
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-
-
?
CO + benzoquinone + H2O
CO2 + benzoquinol
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-
-
?
CO + a quinone + H2O
CO2 + a quinol
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-
-
?
CO + a quinone + H2O
CO2 + a quinol
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the enzyme catalyzes the oxidation of CO to CO2, yielding two electrons and two H+
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-
?
CO + ubiquinone + H2O
CO2 + ubiquinol
ubiquinone is the likely physiological oxidant for CO dehydrogenase
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-
?
CO + ubiquinone + H2O
CO2 + ubiquinol
oxidation of carbon monoxide occurs at the binuclear center with reducing equivalents passed from the redox-active molybdenum to the proximal Fe-S cluster I to the distal Fe-S cluster II and finally to the FAD cofactor
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-
?
additional information
?
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air-stable CO dehydrogenase having a binuclear molybdenum- and copper-containing active site catalyzes the first step in this process, the oxidation of CO to CO2, with the reducing equivalents. Enzyme reduction and reactivity with H2, kinetics, overview
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-
?
additional information
?
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air-stable CO dehydrogenase having a binuclear molybdenum- and copper-containing active site catalyzes the first step in this process, the oxidation of CO to CO2, with the reducing equivalents. Enzyme reduction and reactivity with H2, kinetics, overview
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?
additional information
?
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carbon monoxide dehydrogenases (CO dehydrogenases) are enzymes which catalyze the oxidation of CO to CO2 yielding two electrons and two protons (CO + H2O = CO2 + 2e- + 2H+) or the reverse reaction. CO oxidation by CO dehydrogenase proceeds at a unique bimetallic [CuSMoO2] cluster which matures posttranslationally while integrated into the completely folded apoenzyme
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-
?
additional information
?
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analysis of mechanism of H2 oxidation, which involves initial binding of H2 to the copper of the binuclear center, displacing the bound water, followed by sequential deprotonation through a copper-hydride intermediate to reduce the binuclear center.The enzyme can be reduced by H2 with a limiting rate constant of 5.3/s and a dissociation constant Kd of 0.525 mM, steady-state and stopped-flow rapid reaction kinetics, overview
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-
?
additional information
?
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analysis of mechanism of H2 oxidation, which involves initial binding of H2 to the copper of the binuclear center, displacing the bound water, followed by sequential deprotonation through a copper-hydride intermediate to reduce the binuclear center.The enzyme can be reduced by H2 with a limiting rate constant of 5.3/s and a dissociation constant Kd of 0.525 mM, steady-state and stopped-flow rapid reaction kinetics, overview
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?
additional information
?
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quinones are unusual physiological oxidants for this family of enzymes
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?
additional information
?
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the CO dehydrogenation reaction requires the oxidized state of the enzyme. The oxidation of CO mediated by CO dehydrogenase is followed spectrophotometrically with 1-phenyl-2-(4-iodophenyl)-3-(4-nitrophenyl)-2H-tetrazolium chloride/1-methoxyphenazine methosulfate as artificial electron acceptors. Oxidation of xanthine by CO dehydrogenase
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-
?
additional information
?
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is able to catalyze both the oxidation of CO to CO2 and the oxidation of H2 to protons and electrons
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additional information
?
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is able to catalyze both the oxidation of CO to CO2 and the oxidation of H2 to protons and electrons
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NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
CO + ubiquinone + H2O
CO2 + ubiquinol
ubiquinone is the likely physiological oxidant for CO dehydrogenase
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-
?
additional information
?
-
air-stable CO dehydrogenase having a binuclear molybdenum- and copper-containing active site catalyzes the first step in this process, the oxidation of CO to CO2, with the reducing equivalents. Enzyme reduction and reactivity with H2, kinetics, overview
-
-
?
additional information
?
-
-
air-stable CO dehydrogenase having a binuclear molybdenum- and copper-containing active site catalyzes the first step in this process, the oxidation of CO to CO2, with the reducing equivalents. Enzyme reduction and reactivity with H2, kinetics, overview
-
-
?
additional information
?
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-
carbon monoxide dehydrogenases (CO dehydrogenases) are enzymes which catalyze the oxidation of CO to CO2 yielding two electrons and two protons (CO + H2O = CO2 + 2e- + 2H+) or the reverse reaction. CO oxidation by CO dehydrogenase proceeds at a unique bimetallic [CuSMoO2] cluster which matures posttranslationally while integrated into the completely folded apoenzyme
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?
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
molybdenum-containing cofactor
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the active site molybdenum center located in teh large subunit. The molybdenum becomes reduced in the final step of the reaction
quinone
quinone cofactors interact with CODH at its FAD site
[2Fe-2S]-center
presence of 2 [Fe2S2] clusters, UV-vis spectrum shows a shoulder at 550 nm
[CuSMoO2] cluster
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CO oxidation by CO dehydrogenase proceeds at a unique [Mo+VIO2-S-Cu+I-S-Cys] cluster which matures posttranslationally while integrated into the completely folded apoenzyme. The Mo ion of the cluster is coordinated by the ene-dithiolate of the molybdopterin cytosine dinucleotide cofactor (MCD). The cofactor biosynthesis starts with the MgATP-dependent, reductive sulfuration of [MoVIO3] to [MoVO2SH] which entails the AAA+-ATPase chaperone CoxD. Then MoV is reoxidized and Cu1+-ion is integrated. Copper is supplied by the soluble CoxF protein which forms a complex with the membrane-bound von Willebrand protein CoxE through RGD-integrin interactions and enables the reduction of CoxF-bound Cu2+, employing electrons from respiration. Copper appears as Cu2+-phytate, is mobilized through the phytase activity of CoxF and then transferred to the CoxF putative copperbinding site. The coxG gene does not participate in the maturation of the bimetallic cluster
FAD
FAD is bound in the medium subunit, a flavoprotein
FAD
FAD is bound in the medium subunit. The flavoprotein can be removed from CO dehydrogenase by dissociation with sodium dodecylsulfate, the resulting M(LS)2- or (LS)2-structured CO dehydrogenase species can be reconstituted with the recombinant apoflavoprotein produced in Escherichia coli, structural and functional analysis of FAD binding in CO dehydrogenase
FAD
one noncovalently bound FAD molecule per monomer, FAD-binding occurs on the M subunit and requires conformational changes of subunit M introduced through the binding of subunt M to subunits LS. In air-oxidized CO dehydrogenase, the flavin is fully oxidized
FAD
the GLGTYG sequence, residues 564 to 569, in large subunit CoxL is identical to dinucleotide-binding motif GXGXXG/A, an FAD binding site. The FAD-binding domain of the ferredoxin-NADP+ reductase type is absent
molybdenum cofactor
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presence of a square pyramidal (Mo) oxidized active site, i.e. [(MCD)MoVIOX(Fe-S)CuI(S-Cys)]n, MCD = molybdopterin cytosine dinucleotide, X = OH3 or O4, cofactor reaction mechanism, computational modelling, overview
molybdopterin cofactor
the L subunit carries the molybdenum cofactor, which is a mononuclear complex of Mo and molybdopterin-cytosine dinucleotide (MCD). The latter occurs in a redox state that is reduced by two electrons compared with the fully oxidized state, a tricyclic tetrahydropterin-pyran system. The MCD-molybdenum cofactor is buried at the center of the L subunit and is ligated through a dense network of hydrogen bonds originating from both domains of subunit L. The geometry of the first coordination sphere around the Mo ion is a distorted square pyramid
molybdopterin cofactor
the molybdoprotein of CO dehydrogenase carries the molybdopterin cytosine dinucleotide (MCD)1-type of molybdenum cofactor and the unique active-site loop Gly383-Val-Ala-Tyr-Arg-Cys-Ser-Phe-Arg391, which positions the catalytically essential S-selanylcysteine 388 in a distance of 3.7 A to the molybdenum ion
molybdopterin cofactor
the structure of the active site binuclear center of CO dehydrogenase in its oxidized form, overview. The oxidized Mo(VI) ion has the distorted square-pyramidal coordination geometry seen in other members of the xanthine oxidase family of molybdenum-containing enzymes, with an apical Mo=O and an equatorial plane consisting of a second Mo=O group rather than the catalytically labile Mo-OH seen in other family members and two sulfurs from a pyranopterin cofactor that is common to all molybdenum and tungsten enzymes. The pyranopterin cofactor is present as the dinucleotide of cytosine
additional information
an S-selanylcysteine-containing 88.7-kDa molybdoprotein, a 17.8-kDa iron-sulfur protein, and a 30.2-kDa flavoprotein in a (LMS)2 subunit structure
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additional information
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an S-selanylcysteine-containing 88.7-kDa molybdoprotein, a 17.8-kDa iron-sulfur protein, and a 30.2-kDa flavoprotein in a (LMS)2 subunit structure
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additional information
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presence of FAD, Fe/S clusters, and a [CuSMoO2] coordination in the active site determined by Raman spectra
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additional information
rescue of 50% enzyme activity by in vitro reconstitution of the active site through the supply of sulfide first and subsequently of Cu(I) under reducing conditions. Immature forms of CO dehydrogenase isolated from the bacterium, which are deficient in S and/or Cu at the active site, are similarly activated. The [CuSMoO2] cluster is properly reconstructed. Sulfane sulfur is bound in the active site of CO dehydrogenase. Rebuilding a functional [CuSMoO2] centre by first generating a [MoO3] centre in the active site of CO dehydrogenase
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additional information
-
the enzyme is a molybdo iron-sulfur flavoprotein
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additional information
the enzyme is a molybdo iron-sulfur flavoprotein containing S-selanylcysteine. The redox components of one LMS-structured monomer are the MCD-molybdenum cofactor, composed of a molybdenum ion with two oxo- and one hydroxoligand, complexed by the enedithiolene group of MCD, [2Fe-2S] clusters of type I and type II, and a noncovalently bound FAD molecule
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METALS and IONS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
copper
Cu
Fe-S cluster
proximal Fe-S cluster I and distal Fe-S cluster II
Fe2+
Molybdenum
selenium
[2Fe-2S] cluster
[CuSMoO2] cluster
additional information
Fe2+
-
the small subunit CoxS harbors two [2Fe-2S] iron-sulfur clusters
Molybdenum
in air-oxidized CO dehydrogenase, the oxidation state of Mo is +VI
Molybdenum
Mo/Cu-containing enzyme active site. The overall configuration of the binuclear center is L-MoVIO2-microS-CuI-Cys388, with L representing a bidentate pyranopterin cofactor common to all molybdenum enzymes other than nitrogenase
selenium
an S-selanylcysteine-containing large subunit
selenium
necessity of S-selanylcysteine for the catalyzed reaction, the selenium atom of S-selanylcysteine at the active site is located in a distance of 3.7 A from the Mo ion. It is near the equatorial oxo and hydroxo group of the Mo ion
[2Fe-2S] cluster
a type I and a type II [2Fe-2S] center. The iron-sulfur protein carries the two [2Fe-2S] clusters, which can be distinguished by electron paramagnetic resonance spectroscopy
[2Fe-2S] cluster
the type II 2Fe:2S center is identified in the N-terminal domain and the type I center in the C-terminal domain of the iron-sulfur protein
[2Fe-2S] cluster
two distinct [2Fe-2S] clusters, the small subunit CoxS contains motifs indicative of type I and II [2Fe-2S] cluster, structure and binding strutcures, overview
[2Fe-2S] cluster
two types of [2Fe-2S] clusters, [2Fe-2S] clusters of type I and type II, the two [2Fe-2S] clusters are located in the S subunit. These prosthetic groups form a pathway for the electrons to the FAD. The C-terminal domain (residues 77-161) carries the proximal [2Fe-2S] cluster. The cluster is buried in CO dehydrogenase about 11 A below the protein surface at the interface between the S and the L subunit and is adjacent to the MCD-molybdenum cofactor. The [2Fe-2S] cluster is located at the N terminus of two alpha-helices that participate in a four-helix bundle of twofold symmetry
[2Fe-2S] cluster
two [2Fe-2S] iron-sulfur clusters in the small subunit
[CuSMoO2] cluster
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CO oxidation by CO dehydrogenase proceeds at a unique [Mo+VIO2-S-Cu+I-S-Cys] cluster which matures posttranslationally while integrated into the completely folded apoenzyme. The Mo ion of the cluster is coordinated by the ene-dithiolate of the molybdopterin cytosine dinucleotide cofactor (MCD). The cofactor biosynthesis starts with the MgATP-dependent, reductive sulfuration of [MoVIO3] to [MoVO2SH] which entails the AAA+-ATPase chaperone CoxD. Then MoV is reoxidized and Cu1+-ion is integrated. Copper is supplied by the soluble CoxF protein which forms a complex with the membrane-bound von Willebrand protein CoxE through RGD-integrin interactions and enables the reduction of CoxF-bound Cu2+, employing electrons from respiration. Copper appears as Cu2+-phytate, is mobilized through the phytase activity of CoxF and then transferred to the CoxF putative copperbinding site. The coxG gene does not participate in the maturation of the bimetallic cluster
[CuSMoO2] cluster
the Mo-ion in the oxidized cluster is in +VI oxidation state and upon incubation with CO or sodium dithionite is reduced to Mo(IV). The Cu ion permanently remains in the +1 oxidation state. The ligands around Mo form a distorted square pyramidal geometry. The large subunit forms a molybdoprotein
additional information
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metal contents are determined by inductively coupled plasma atomic emission spectrometry
additional information
the binuclear active site contains copper as well as molybdenum
additional information
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the binuclear active site contains copper as well as molybdenum
additional information
the removal of Cu and S from the active site changes the functional [CuSMoO2] centre into a non-functional [MoO3] centre. The insertion of a sulfur atom from sodium sulfide into the [MoO3] center yielding a [MoO2S] center. The latter does not catalyze the oxidation of CO referring to a nonfunctional Mo-centre. Resulfuration of the [MoO3] centre and transfer of Cu from the Cu(I)thiourea complex to the [MoO2S] centre partially restores the specific CO oxidizing activity
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
thiol inhibition of CO dehydrogenase
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KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
additional information
additional information
-
rapid reaction kinetics, steady-state kinetics
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TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
51.1
CO
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pH 7.2, 25°C, reductive half-reaction of enzyme CODH with CO
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
2.6
recombinant purified enzyme, reconstitution of the active site with sulfur and copper, pH 7.2, 30°C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
7.2
pH RANGE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
5.5 - 10
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only modest loss of activity at these extreme pHs indicates that ionization of functional groups in the active site is not as critical to catalysis for CODH
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
25
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
genes coxL, coxM, and coxS; formerly Pseudomonas carboxydovorans strain OM5, genes coxS, coxM, and coxL
UniProt
genes coxL, coxM, and coxS; formerly Pseudomonas carboxydovorans strain OM5, genes coxS, coxM, and coxL encoded on the low-copy-number 133,058 bp-circular DNA megaplasmid pHCG3
UniProt
genes coxL, coxM, and coxS; genes coxS, coxM, and coxL
UniProt
genes coxL, coxM, and coxS; the enzyme is encoded by coxMSL structural genes in the megaplasmid-localized coxBCMSLDEFGHIK gene cluster
UniProt
large, medium, and small subunits; large, medium, and small subunits of CODH are encoded by the structural genes coxL, coxM, and coxS, respectively. They reside on the 128-kb megaplasmid pHCG3
UniProt
P19921 i.e. small subunit CoxS, P19920 i.e. medium subunit CoxM, P19919 i.e. large subunit CoxL
UniProt
SOURCE TISSUE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
SOURCE
additional information
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Oligotropha carboxidovorans is aerobe and able to grow with CO as sole source of both carbon and energy
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY
GeneOntology No.
LITERATURE
SOURCE
-
role of the pleckstrin homology domain of enzyme CoxG in recruiting CO dehydrogenase to the cytoplasmic membrane enabling electron transfer from the enzyme to the respiratory chain
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
evolution
malfunction
metabolism
physiological function
additional information
evolution
CO dehydrogenase is a member of the xanthine oxidase family
evolution
CO dehydrogenase is a prototype of the molybdenum hydroxylase sequence family
evolution
despite the unique nature of the binuclear active site of CO dehydrogenase the enzyme is clearly a member of the xanthine oxidase family of molybdenum-containing enzymes
evolution
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the enzyme belongs to the molybdenum hydroxylase (xanthine oxidase) family of Mo enzymes
evolution
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the enzyme belongs to the noncanonical members of the xanthine oxidase family. The Mo-containing CO dehydrogenase from Oligotropha carboxidovorans and related organisms is distinct from the highly O2-sensitive Ni/Fe-containing CO dehydrogenase from obligate anaerobes such as Clostridum thermoaceticum or Methanosarcina barkerii. Quinones are unusual physiological oxidants for this family of enzymes, the overall fold of the FAD-containing domain of CO dehydrogenase resembles the dehydrogenase rather than the oxidase form of the bovine xanthine oxidoreductase, particularly with regard to the position of the mobile loop referred to above that is involved in the Dto-O conversion, but there are significant differences in the environment of the FAD in CO dehydrogenase and xanthine dehydrogenase. A Lys-Asp pair near the pyrimidine subnucleus of the flavin is preserved, for example, but the positions of the Ile and aromatic residues are reversed, with the Ile on the re side and Tyr (a Phe in the bovine enzyme) on the si side of the isoalloxazine ring
evolution
the enzyme is a member of the xanthine oxidase (XO) family of pyranopterin molybdenum enzymes that typically catalyse the oxidative hydroxylation of N-heterocyle and aldehyde substrates
evolution
the Mo- and Cu-containing CO dehydrogenase from Oligotropha carboxydovorans is both mechanistically and structurally distinct from the extremely O2-sensitive Ni- and Fe-containing CO dehydrogenase from organisms such as Moorella thermoacetica or Methanosarcina barkerii. On the basis of overall architecture and sequence homology, the Mo/Cu CO dehydrogenase belongs to the xanthine oxidase family of enzymes but is unique among members of this large and broadly distributed family in several regards: the reaction catalyzed is not formally a hydroxylation reaction involving hydride abstraction from substrate. The enzyme utilizes ubiquinone as the oxidizing substrate rather than O2 or NADas oxidizing substrate, and, most significantly, its unique binuclear active site contains copper as well as molybdenum
malfunction
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metal cluster composition, structure and function of CO dehydrogenase synthesized in mutants of Oligotropha carboxidovorans strain OM5 in which the genes coxE, coxF and coxG are disrupted by insertional mutagenesis, recombinant expression in Escherichia coli strain S17-1, overview. Mutants in coxG retain the ability to utilize CO, although at a lower growth rate. They contain a regular CO dehydrogenase with a functional catalytic site. Disruption of coxD leads to a phenotype of D-km which is impaired in the utilization of CO, whereas the utilization of H2 plus CO2 is not affected. The deletion of coxG leads to a phenotype which is still able to utilize CO, although the generation time increases considerably from 21 h (wild-type) to 149 h. Under appropriate induction conditions, bacteria synthesize a fully assembled apo-CO dehydrogenase, which cannot oxidize CO. Apo-CO dehydrogenase contains a [MoO3] site in place of the [CuSMoO2] clusters
malfunction
the removal of Cu and S from the active site changes the functional [CuSMoO2] centre into a non-functional [MoO3] centre
malfunction
thiol inhibition of CO dehydrogenase may be a physiologically important mechanism of enzyme regulation
metabolism
four other genes (coxB, coxC, coxH and coxK) are predicted to encode proteins possessing one (CoxB) to as many as nine (CoxK) transmembrane helices, one or more of which are likely to be involved in anchoring CO dehydrogenase to its physiological position on the inner side of the cytoplasmic membrane
metabolism
-
the CoxD protein is a distinct AAA+ ATPase. CoxD operates in the maturation of the CO dehydrogenase bimetallic cluster, particularly in the sulfuration of the [MoO3]-site and in ATP-dependent chaperone function. The genes coxE and coxF are both obligatory for the utilization of CO as a growth substrate
metabolism
-
the enzyme catalyzes the critical first step in this process, the oxidation of CO to CO2 with the reducing equivalents thus obtained ultimately being passed on ultimately to a CO-insensitive terminal oxidase
physiological function
-
carbon monoxide dehydrogenases are key to the generation of a proton motive force across the cytoplasmic membrane for ATP synthesis or cooperate with acetyl-CoA synthase in the biosynthesis of acetyl-CoA
physiological function
-
Oligotropha carboxidovorans is a carboxydotrophic bacterium capable of aerobic, chemolithoautotrophic growth using COas a sole source of carbon and energy. The key enzyme involved in this facultative metabolism is an air-stable molybdenum-containing CO dehydrogenase that catalyzes the oxidation of CO to CO2
physiological function
-
the enzyme catalyzes the oxidation of CO to CO2, thereby providing carbon and energy to the organism and maintaining subtoxic levels of CO in the troposphere
physiological function
the enzyme is a molybdenum-containing iron-sulfur flavoprotein and is the key enzyme in the chemolithoautotrophic utilization of CO by Oligotropha carboxidovorans strain OM5. Conserved protein building blocks constitute CODH and the other molybdenum hydroxylases
additional information
mechanism of Mo/Cu carbon monoxide dehydrogenase, electronic structure contributions to reactivity, overview
additional information
-
the enzyme has a unique heterobimetallic Mo/Cu active site, mass spectrometric and EPR spectra analysis, overview. Key to the catalytic mechanism of the CODH site is the electronic communication between the Mo and Cu atoms
additional information
-
the enzyme is noncanonical in terms of the structure of the molybdenum center, the nature of the reaction catalyzed, the type of redox-active centers that are found, or some combination of these. The active site is located in the large subunit
additional information
the enzyme possesses a deeply buried binuclear center of CO dehydrogenase activity
additional information
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the enzyme possesses a deeply buried binuclear center of CO dehydrogenase activity
additional information
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
additional information
-
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
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
17800
(alphabetagamma)2, 2 * 88700, large subunit, + 2 * 30200, medoum subunit, + 2 * 17800, small subunit, SDS-PAGE
30000
-
(alphabetagamma)2, 2 * 89000, large subunit, + 1 * 30000, medium subunit, + 1 * 1800, small subunit, SDS-PAGE
30200
(alphabetagamma)2, 2 * 88700, large subunit, + 2 * 30200, medoum subunit, + 2 * 17800, small subunit, SDS-PAGE
41000
recombinant monomeric deflavo medium subunit, gel filtration
88700
(alphabetagamma)2, 2 * 88700, large subunit, + 2 * 30200, medoum subunit, + 2 * 17800, small subunit, SDS-PAGE
89000
-
(alphabetagamma)2, 2 * 89000, large subunit, + 1 * 30000, medium subunit, + 1 * 1800, small subunit, SDS-PAGE
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
heterohexamer
additional information
heterohexamer
(abc)2 structure, each protomer of the enzyme has a small subunit (CoxS, 18 kDa) with two [2Fe-2S] iron-sulfur clusters, a medium subunit (CoxM, 30 kDa) that possesses FAD, and a large subunit (CoxL, 89 kDa) that has the active site binuclear center
heterohexamer
(alphabetagamma)2 hexamer, with a large subunit (coxL, 88.7 kDa) containing the binuclear active site, a medium subunit (coxM, 30.2 kDa) with FAD, and a small subunit (coxS, 30.2 kDa) containing two spinach ferredoxin-like [2Fe-2S] clusters
heterohexamer
(alphabetagamma)2, 2 * 88700, large subunit, + 2 * 30200, medoum subunit, + 2 * 17800, small subunit, SDS-PAGE
heterohexamer
-
(alphabetagamma)2, 2 * 89000, large subunit, + 1 * 30000, medium subunit, + 1 * 1800, small subunit, SDS-PAGE
heterohexamer
CO dehydrogenase is composed of a 88.7 kDa molybdoprotein (L subunit), a 30.2 kDa flavoprotein (M subunit), and a 17.8 kDa iron-sulfur protein (S subunit) in a (LMS)2 subunit composition
heterohexamer
CO dehydrogenase is composed of an 88.7-kDa molybdoprotein (subunit L), a 30.2-kDa flavoprotein (subunit M), and a 17.8-kDa iron-sulfur protein (subunit S). It is organized as a dimer of LMS heterotrimers
additional information
the enzyme is an S-selanylcysteine-containing 88.7-kDa molybdoprotein, a 17.8-kDa iron-sulfur protein, and a 30.2-kDa flavoprotein in a (LMS)2 subunit structure
additional information
-
the enzyme is an S-selanylcysteine-containing 88.7-kDa molybdoprotein, a 17.8-kDa iron-sulfur protein, and a 30.2-kDa flavoprotein in a (LMS)2 subunit structure
additional information
-
the the active site molybdenum center is located in the large subunit, while the medium subunit contains FAD, and the small subunit contains the [2Fe-2S]-clusters
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
enzyme in complex with inhibitor n-butylisonitrile, PDB ID 1N62, structure analysis
purified reconstituted enzyme, hangig drop vapour diffusion method, X-ray diffraction structure determination and analysis at 1.7 A resolution
vapor diffusion method using 0.8 M KH2PO4, 0.8 M NaH2PO4, 2% MPD, and 100 mM HEPES, pH 7.3, crystals containing cyanide are cocrystallized in the presence of 4 mM potassium cyanide, X-ray diffraction structure determination and analysis at 2.36-2.5 A resolution
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
additional information
additional information
-
metal cluster composition, structure and function of CO dehydrogenase synthesized in mutants of Oligotropha carboxidovorans strain OM5 in which the genes coxE, coxF and coxG are disrupted by insertional mutagenesis, recombinant expression in Escherichia coli strain S17-1, overview. Mutants in coxG retain the ability to utilize CO, although at a lower growth rate. They contain a regular CO dehydrogenase with a functional catalytic site. The CoxD protein is a distinct AAA+ ATPase. CoxD operates in the maturation of the CO dehydrogenase bimetallic cluster, particularly in the sulfuration of the [MoO3]-site and in ATP-dependent chaperone function. Disruption of coxD leads to a phenotype of D-km which is impaired in the utilization of CO, whereas the utilization of H2 plus CO2 is not affected. Under appropriate induction conditions, bacteria synthesize a fully assembled apo-CO dehydrogenase, which cannot oxidize CO. Apo-CO dehydrogenase contained a [MoO3] site in place of the [CuSMoO2] clusters. The genes coxE and coxF are both obligatory for the utilization of CO as a growth substrate
additional information
the flavoprotein can be removed from CO dehydrogenase by dissociation with sodium dodecylsulfate, the resulting M(LS)2- or (LS)2-structured CO dehydrogenase species can be reconstituted with the recombinant apoflavoprotein produced in Escherichia coli. Binding of FAD to the reconstituted deflavo (LMS)2 species occurs with second-order kinetics and high affinity. 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 is disordered. The structural changes related to heterotrimeric complex formation or FAD binding are transmitted to the iron-sulfur protein, structural and functional analysis of FAD binding in CO dehydrogenase
additional information
-
the flavoprotein can be removed from CO dehydrogenase by dissociation with sodium dodecylsulfate, the resulting M(LS)2- or (LS)2-structured CO dehydrogenase species can be reconstituted with the recombinant apoflavoprotein produced in Escherichia coli. Binding of FAD to the reconstituted deflavo (LMS)2 species occurs with second-order kinetics and high affinity. 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 is disordered. The structural changes related to heterotrimeric complex formation or FAD binding are transmitted to the iron-sulfur protein, structural and functional analysis of FAD binding in CO dehydrogenase
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant protein, purification by Ni-NTA and size exclusion chromatography
recombinant recombinant M subunit 8fold by ultracentrifugation, anion exchange chromatography, and gel filtration from Escherichia coli strain BL21(DE3)
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
genes coxS, coxM, and coxL, recombinant expression of FAD-reconstituted enzyme and of monomeric deflavo medium subunit in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain DH5alpha
large, medium, and small subunits of CODH are encoded by the structural genes coxL, coxM, and coxS, respectively. They reside on a 128-kb megaplasmid pHCG3, DNA and amino acid sequence determination and analysis, and molecular organization of the coxMSL gene cluster, sequence comparisons
the enzyme is encoded by the coxMSL structural genes in the megaplasmid-localized coxBCMSLDEFGHIK gene cluster
the enzyme is encoded by the megaplasmid-localized coxBCMSLDEFGHIK gene cluster, the cosMSL structural genes encoding the enzyme
-
RENATURED/Commentary
ORGANISM
UNIPROT
LITERATURE
reconstitution of 50% enzyme activity by in vitro reconstitution of the active site through the supply of sulfide first and subsequently of Cu(I) under reducing conditions. Immature forms of CO dehydrogenase isolated from the bacterium, which are deficient in S and/or Cu at the active site, are similarly activated. The [CuSMoO2] cluster is properly reconstructed
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
synthesis
establishment of a functional heterologous expression system of Moco-free apo-CODH in Escherichia coli. The expression of the CoxMSL genes alone results in a colorless and inactive protein that lacks the cofactors. Expression of an active protein requires coexpression of CoxI
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Schubel, U.; Kraut, M.; Morsdorf, G.; Meyer, O.
Molecular characterization of the gene cluster coxMSL encoding the molybdenum-containing carbon monoxide dehydrogenase of Oligotropha carboxidovorans
J. Bacteriol.
177
2197-2203
1995
Afipia carboxidovorans (P19919 and P19920 and P19921), Afipia carboxidovorans, Afipia carboxidovorans OM5 (P19919 and P19920 and P19921), Afipia carboxidovorans OM5
Dobbek, H.; Gremer, L.; Meyer, O.; Huber, R.
Crystal structure and mechanism of CO dehydrogenase, a molybdo iron-sulfur flavoprotein containing S-selanylcysteine
Proc. Natl. Acad. Sci. USA
96
8884-8889
1999
Afipia carboxidovorans (P19919 and P19920 and P19921)
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.
133
12934-12936
2011
Afipia carboxidovorans
Zhang, B.; Hemann, C.F.; Hille, R.
Kinetic and spectroscopic studies of the molybdenum-copper CO dehydrogenase from Oligotropha carboxidovorans
J. Biol. Chem.
285
12571-12578
2010
Afipia carboxidovorans
Stein, B.; Kirk, M.
Orbital contributions to CO oxidation in Mo-Cu carbon monoxide dehydrogenase
Chem. Commun. (Camb.)
50
1104-1106
2014
Hydrogenophaga pseudoflava (P19913 and P19914 and P19915), Afipia carboxidovorans (P19920)
Gourlay, C.; Nielsen, D.; White, J.; Knottenbelt, S.; Kirk, M.; Young, C.
Paramagnetic active site models for the molybdenum-copper carbon monoxide dehydrogenase
J. Am. Chem. Soc.
128
2164-2165
2006
Afipia carboxidovorans
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.
275
1864-1872
2000
Afipia carboxidovorans (P19919 and P19920 and P19921), Afipia carboxidovorans, Afipia carboxidovorans DSM 1227 (P19919 and P19920 and P19921)
Wilcoxen, J.; Hille, R.
The hydrogenase activity of the molybdenum/copper-containing carbon monoxide dehydrogenase of Oligotropha carboxidovorans
J. Biol. Chem.
288
36052-36060
2013
Afipia carboxidovorans (P19919 and P19920 and P19921), Afipia carboxidovorans
Resch, M.; Dobbek, H.; Meyer, O.
Structural and functional reconstruction in situ of the [CuSMoO 2] active site of carbon monoxide dehydrogenase from the carbon monoxide oxidizing eubacterium Oligotropha carboxidovorans
J. Biol. Inorg. Chem.
10
518-528
2005
Afipia carboxidovorans (P19919 and P19920 and P19921), Afipia carboxidovorans DSM 1227 (P19919 and P19920 and P19921)
Pelzmann, A.; Mickoleit, F.; Meyer, O.
Insights into the posttranslational assembly of the Mo-, S- and Cu-containing cluster in the active site of CO dehydrogenase of Oligotropha carboxidovorans
J. Biol. Inorg. Chem.
19
1399-1414
2014
Afipia carboxidovorans
Hille, R.; Dingwall, S.; Wilcoxen, J.
The aerobic CO dehydrogenase from Oligotropha carboxidovorans
J. Biol. Inorg. Chem.
20
243-251
2015
Afipia carboxidovorans (P19919 and P19920 and P19921), Afipia carboxidovorans
Kaufmann, P.; Duffus, B.R.; Teutloff, C.; Leimkuehler, S.
Functional Studies on Oligotropha carboxidovorans molybdenum-copper CO dehydrogenase produced in Escherichia coli
Biochemistry
57
2889-2901
2018
Afipia carboxidovorans (P19921 and P19920 and P19919), Afipia carboxidovorans, Afipia carboxidovorans DSM 1227 (P19921 and P19920 and P19919)
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