Identification of a cysteine involved in the interaction between carbon monoxide dehydrogenase and corrinoid/Fe‐S protein from <i>Clostridium thermoaceticum</i>

Shanmugasundaram, Thangavel; Sundaresh, C.S.; Kumar, Ganesh K.

doi:10.1016/0014-5793(93)81808-d

Cited by 4 publications

(2 citation statements)

References 20 publications

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“…During catalysis, we propose that the CFeSP forms complexes with CODH/ACS and MeTr. Evidence for the CODH/ACS-CFeSP complex in C. thermoaceticum is based on their coelution under some chromatographic conditions (8,39) and the identification of a specific cysteine residue in the R subunit that can form a disulfide link between the two proteins (39). In methanoarchaea, the interaction between these proteins is even tightersa fiveprotein complex that has CODH, ACS, CFeSP, and MeTr activities has been isolated from Ms. thermophila (40) and Ms. barkeri (41)(42)(43).…”

Section: Discussionmentioning

confidence: 99%

Role of the [4Fe-4S] Cluster in Reductive Activation of the Cobalt Center of the Corrinoid Iron−Sulfur Protein from Clostridium thermoaceticum during Acetate Biosynthesis

Menon

Ragsdale

1998

Biochemistry

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The corrinoid iron-sulfur protein (CFeSP) from Clostridium thermoaceticum functions as a methyl carrier in the Wood-Ljungdahl pathway of acetyl-CoA synthesis. The small subunit (33 kDa) contains cobalt in a corrinoid cofactor, and the large subunit (55 kDa) contains a [4Fe-4S] cluster. The cobalt center is methylated by methyltetrahydrofolate (CH3-H4folate) to form a methylcobalt intermediate and, subsequently, is demethylated by carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS). The work described here demonstrates that the [4Fe-4S] cluster is required to facilitate the reactivation of oxidatively inactivated Cob(II)amide to the active Co(I) state. Site-directed mutagenesis of the large subunit gene was used to change residue 20 from cysteine to alanine, which resulted in formation of a cluster with EPR and redox properties consistent with those of [3Fe-4S] clusters. The midpoint potential of the cluster in the C20A variant was approximately 500 mV more positive than that of the [4Fe-4S] cluster in the native enzyme. Accordingly, it was found that the Co center in the C20A mutant protein could be reduced artificially but was severely crippled in its ability to be reduced by physiological electron donors. This is probably because the reduced cluster of the C20A protein cannot provide the driving force needed to reduce Co(II) to Co(I), since the Co(II/I) midpoint potential is -504 mV. The C20A variant also was unable to catalyze the steady-state synthesis of acetyl-CoA when CH3-H4folate or methyl iodide were provided as methyl donors and CO and CODH/ACS as reductants. Addition of chemical reductants rescued the catalytically crippled variant form in both of these reactions. On the other hand, in single-turnover reactions, the methyl-Co state of the altered protein was fully active in methylating H4folate and in synthesizing acetyl-CoA in the presence of CO and CoA. The combined results strongly indicate that the FeS cluster of the CFeSP is necessary for reductive activation of Co(II) to Co(I) by physiological reductants but is not required for catalysis, e.g., demethylation of CH3-H4folate or methylation of CODH/ACS. We propose that, during reductive activation, electrons flow from the reduced electron-transfer protein (e.g., CODH/ACS or reduced ferredoxin (Fd)) to the FeS cluster which then directs electrons to the cobalt center for catalysis. These results also support earlier hypotheses that the methylation and demethylation reactions involving the CFeSP are SN2-type nucleophilic displacement reactions and do not involve radical chemistry.

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Section: Discussionmentioning

confidence: 99%

Role of the [4Fe-4S] Cluster in Reductive Activation of the Cobalt Center of the Corrinoid Iron−Sulfur Protein from Clostridium thermoaceticum during Acetate Biosynthesis

Menon

Ragsdale

1998

Biochemistry

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“…Two of the other proteins required for this activity bind to a. The corrinoid/iron-sulfur enzyme, which functions to transfer the methyl group to CODH, appears to be linked in-vivo to a cysteine-506 of CODH via a disulfide bond (Shanmugasundaram et al, 1993). The ferredoxin that stimulates acetyl-CoA synthesis appears to dock at residues 229-239 of the a subunit of CODH (Shanmugasundaram & Wood, 1992).…”

Section: Discussionmentioning

confidence: 99%

Decomposition of Carbon Monoxide Dehydrogenase into .alpha. Metallosubunits and a Catalytically-Active Form Consisting Primarily of .beta. Metallosubunits

Xia

Lindahl²

1995

Biochemistry

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Carbon monoxide dehydrogenase from Clostridium thermoaceticum, with an alpha 3 beta 3 quaternary structure, was incubated with dithiothreitol and 740-78,000 equiv/alpha 3 beta 3 of sodium dodecyl sulfate (SDS), followed by electrophoresis under anaerobic native conditions. Three catalytically-active bands and four inactive bands were obtained, in proportions dependent on the amount of SDS added. The catalytically-active bands, called SM-CODH, NM-CODH, and FM-CODH, migrated slower than, similar to, and faster than native enzyme, respectively. Two-dimensional electrophoresis revealed that SM-CODH and NM-CODH contained approximately equal proportions of the alpha and beta subunits, while the beta/alpha ratio for FM-CODH was about 2.7. The four inactive bands were identified as the beta subunit, two forms of the alpha subunit (called alpha and alpha'), and a form that migrated similarly to native enzyme. Overloaded gels revealed that alpha and each active band had brown color, indicating intact Fe-S clusters in these species. Higher concentrations of SDS (> 1600 equiv/alpha 3 beta 3) and/or incubation at temperatures > 15 degrees C yielded more alpha and beta subunits at the expense of the catalytically-active bands. When incubated at 70 degrees C in 78,000 equiv/alpha 3 beta 3 of SDS, alpha quantitatively converted to alpha', suggesting that alpha' is a denatured form. FM-CODH appears to be an intermediate in the decomposition of CODH by SDS and to have either an alpha 1 beta 3 or alpha 1 beta 2 quaternary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

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Carbon-monoxide dehydrogenase (ferredoxin)

2009

Class 1 · Oxidoreductases

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Identification of a cysteine involved in the interaction between carbon monoxide dehydrogenase and corrinoid/Fe‐S protein from Clostridium thermoaceticum

Cited by 4 publications

References 20 publications

Role of the [4Fe-4S] Cluster in Reductive Activation of the Cobalt Center of the Corrinoid Iron−Sulfur Protein from Clostridium thermoaceticum during Acetate Biosynthesis

Role of the [4Fe-4S] Cluster in Reductive Activation of the Cobalt Center of the Corrinoid Iron−Sulfur Protein from Clostridium thermoaceticum during Acetate Biosynthesis

Decomposition of Carbon Monoxide Dehydrogenase into .alpha. Metallosubunits and a Catalytically-Active Form Consisting Primarily of .beta. Metallosubunits

Carbon-monoxide dehydrogenase (ferredoxin)

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