A Methylnickel Intermediate in a Bimetallic Mechanism of Acetyl-Coenzyme A Synthesis by Anaerobic Bacteria

Kumar, Manoj; Qiu, Di; Spiro, Thomas G.; Ragsdale, Stephen W.

doi:10.1126/science.270.5236.628

Cited by 68 publications

(59 citation statements)

References 37 publications

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“…Given the prevalence of hydrogenase-and urease-associated accessory functions and the similarities between the Ni-CODHs analyzed to date, we expect that cooCTJ analogs will be found associated with all other Ni-CODH systems. Indeed, the cooCTJ functions may represent a minimum accessory complement if, for example, the Clostridium thermoaceticum CODH C center, site of CO oxidation, and A center, site of acetyl coenzyme A synthesis (33), require distinct Ni-insertion functions.…”

Section: Discussionmentioning

confidence: 99%

In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ

1997

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The products of cooCTJ are involved in normal in vivo Ni insertion into the carbon monoxide dehydrogenase (CODH) of Rhodospirillum rubrum. Located on a 1.5-kb DNA segment immediately downstream of the CODH structural gene (cooS), two of the genes encode proteins that bear motifs reminiscent of other (urease and hydrogenase) Ni-insertion systems: a nucleoside triphosphate-binding motif near the N terminus of CooC and a run of 15 histidine residues regularly spaced over the last 30 amino acids of the C terminus of CooJ. A Gm r ⍀-linker cassette was developed to create both polar and nonpolar (60 bp) insertions in the cooCTJ region, and these, along with several deletions, were introduced into R. rubrum by homologous recombination. Analysis of the exogenous Ni levels required to sustain CO-dependent growth of the R. rubrum mutants demonstrated different phenotypes: whereas the wild-type strain and a mutant bearing a partial cooJ deletion (of the region encoding the histidine-rich segment) grew at 0.5 M Ni supplementation, strains bearing Gm r ⍀-linker cassettes in cooT and cooJ required approximately 50-fold-higher Ni levels and all cooC insertion strains, bearing polar or nonpolar insertions, grew optimally at 550 M Ni.The phototrophic bacterium Rhodospirillum rubrum induces synthesis of an Ni-and Fe-containing carbon monoxide dehydrogenase (CODH) upon anaerobic exposure to CO (3, 4) and couples CO oxidation to H 2 evolution as a source of energy (31). Limited evidence suggests that accessory functions are necessary for posttranslational Ni insertion and CODH activity in R. rubrum: an Ni-deficient apo-CODH is found in induced cultures deprived of Ni; substantially higher Ni concentrations are required for activation of apo-CODH in vitro than in vivo; and there is an elevated Ni requirement for CO-dependent growth of a mutant bearing an insertion in cooC, a gene immediately downstream of the CODH structural gene (5,13,31).Aside from the physiology of Ni insertion, the R. rubrum CODH has been well characterized biochemically (4, 14), genes encoding its synthesis and several additional proteins likely involved in H 2 production have been cloned and sequenced (16,17,30), active CODH has been heterologously expressed (24), and the CO-responsive transcriptional regulator is under investigation (24, 53). The hydrogenase (CooH) and CODH (CooS) genes occur in two operons, cooMKLXUH and cooFSCTJ, whose coding regions are separated by a 450-bp interval, with the CO-responsive transcriptional activator (CooA) encoded 137 nucleotides downstream of cooJ.Formation of the Ni-containing center(s) of other Ni-CODH enzymes has not been characterized, although the activities are prevalent in diverse anaerobic archaea and bacteria (15). Both the spectroscopic similarities of purified enzymes (26) and evident conservation of a limited number of potential metal ligands in an alignment of bacterial and archaeal CODH sequences (e.g., see reference 30) are consistent with conservation of structure and function of the Ni-containing C center...

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Section: Discussionmentioning

confidence: 99%

In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ

1997

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“…Single Turnover Kinetics of the Variant Methylated CFeSPWhen the wild type methylated CFeSP is reacted with CODH/ ACS, a low potential metal center (cluster A) on ACS undergoes methylation as the CFeSP is converted to the Co(I) state (25). It remained a possibility that there is some degree of radical chemistry in this reaction (see below for discussion).…”

Section: [4fe-4s] Cluster Requirement For B 12 Reductive Activationmentioning

confidence: 99%

The Role of an Iron-Sulfur Cluster in an Enzymatic Methylation Reaction

Menon¹,

Ragsdale

1999

Journal of Biological Chemistry

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This paper focuses on how a methyl group is transferred from a methyl-cobalt(III) species on one protein (the corrinoid iron-sulfur protein (CFeSP)) to a nickel iron-sulfur cluster on another protein (carbon monoxide dehydrogenase/acetyl-CoA synthase). This is an essential step in the Wood-Ljungdahl pathway of anaerobic CO and CO 2 fixation. The results described here strongly indicate that transfer of methyl group to carbon monoxide dehydrogenase/acetyl-CoA synthase occurs by an S N 2 pathway. They also provide convincing evidence that oxidative inactivation of Co(I) competes with methylation. Under the conditions of our anaerobic assay, Co(I) escapes from the catalytic cycle one in every 100 turnover cycles. Reductive activation of the CFeSP is required to regenerate Co(I) and recruit the protein back into the catalytic cycle. Our results strongly indicate that the [4Fe-4S] cluster of the CFeSP is required for reductive activation. They support the hypothesis that the [4Fe-4S] cluster of the CFeSP does not participate directly in the methyl transfer step but provides a conduit for electron flow from physiological reductants to the cobalt center.

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“…Examples include nitrile hydratase [1][2][3][4][5][6][7][8][9][10] and the A-cluster of carbon monoxide dehydrogenase/acetyl CoA synthase [11][12][13][14][15][16][17][18][19][20][21][22][23]. Understanding the catalytic mechanisms of these enzymes can be greatly aided by the study of small-molecule analogs, or model complexes, which reproduce the active-site structure and/or the function of the enzyme.…”

Section: Introductionmentioning

confidence: 99%

Incorporation of thiolate donation using 2,2′-dithiodibenzaldehyde: Complexes of a pentadentate N2S3 ligand with relevance to the active site of Co nitrile hydratase

Smucker

VanStipdonk

Eichhorn

2007

Journal of Inorganic Biochemistry

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The use of 2,2′-dithiodibenzaldehyde (DTDB) as a reactant for incorporating thiolate donors into the coordination sphere of a transition metal complex without the need for protecting groups is expanded to include the synthesis of complexes with pentadentate ligands. The ligand N,N′-bis (thiosalicylideneimine)-2,2′-thiobis(ethylamine) (tsaltp) is synthesized at a cobalt center by the reaction of DTDB with a Co complex of thiobis(ethylamine). The resulting Co complexes are thus coordinated by the N 2 S 3 pentadentate ligand through two imine N atoms, two thiolate S atoms, and one thioether S atom. A dimeric, bis-thiolate-bridged complex (1) is isolated and converted to a monomeric CN adduct (2) by treatment with KCN. The N 2 S 3 coordination environment provided by the tsaltp ligand is similar to that provided by the protein donors at the active site of the nitrile hydratase enzymes, with 2 being the first octahedral Co complex reported with such a coordination sphere.

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A Methylnickel Intermediate in a Bimetallic Mechanism of Acetyl-Coenzyme A Synthesis by Anaerobic Bacteria

Cited by 68 publications

References 37 publications

In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ

In vivo nickel insertion into the carbon monoxide dehydrogenase of Rhodospirillum rubrum: molecular and physiological characterization of cooCTJ

The Role of an Iron-Sulfur Cluster in an Enzymatic Methylation Reaction

Incorporation of thiolate donation using 2,2′-dithiodibenzaldehyde: Complexes of a pentadentate N2S3 ligand with relevance to the active site of Co nitrile hydratase

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