Abstract
Acetogenic bacteria, as determined with Clostridium thermoaceticum, synthesize acetate by the acetyl-CoA pathway which involves the reduction of CO2 to a methyl group and then combination of the methyl with CoA and a carbonyl group formed from CO or CO2 (Wood, H.G., Ragsdale, S.W., and Pezacka, E. (1986) Trends Biochem. Sci. 11, 14-18). Carbon monoxide dehydrogenase (CODH), the key enzyme in this pathway not only catalyzes the oxidation of CO to CO2 but also the final step, the synthesis of acetyl-CoA from a methyl group, CO, and CoA. Previously, it has been shown that ferredoxin can stimulate exchange of CO with CH3 14COSCoA (Ragsdale, S.W., and Wood, H.G. (1985) J. Biol. Chem. 260, 3970-3977). In the present study, it has been observed that ferredoxin and CODH can form an electrostatically stabilized complex. In order to identify the ferredoxin binding region on CODH, the ferredoxin and CODH were cross-linked by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The cross-linked CODH-ferredoxin adduct was enzymatically as active as the uncross-linked complex. The native CODH and cross-linked CODH-ferredoxin complex were subjected to cyanogen bromide cleavage. By comparison of the high-performance liquid chromatography peptide profiles, it was observed that the mobility of at least one peptide is altered in the CODH-ferredoxin cross-linked complex. The peptide was identified with residues 229-239 of the alpha-subunit of CODH.
Highlights
Thermoaceticum, synthesize acetateby the acetyl-coA pathway which involvesthe reduction of COz to a methyl group and combination of the methyl with
In the presence of carbonmonoxide dehydrogenase (CODH) and ferredoxin (l:l)n,o ferredoxin could be detected passing through the membrane at low ionic strength, consistent with the hypothesis that all of the ferredoxin was complexed to the dehydrogenase
In order to determine the stoichiometry of ferredoxin-CODH complex, CODH was incubated with various levels of ferredoxin and subjected to membrane filtration and the bound ferredoxin was measured (Fig. 1).By this method, the stoichiometry of the CODHferredoxin complex wascalculated to be 1:l.The dependence of ionic strength on the complex formation was tested (Fig. 2)
Summary
Complex Formation between CODH and Ferredoxin-Membrane ultrafiltration (Table I) experiments provided evidence ofcomplex formation betweenCODH and ferredoxin. As expected for a 7,000-kDa protein, ferredoxin alone passed readily through the membrane filter (YM-30,M,30,000 cutoff) at either low or high ionic strengths. In the presence of CODH and ferredoxin (l:l)n,o ferredoxin could be detected passing through the membrane at low ionic strength, consistent with the hypothesis that all of the ferredoxin was complexed to the dehydrogenase. In order to determine the stoichiometry of ferredoxin-CODH complex, CODH was incubated with various levels of ferredoxin and subjected to membrane filtration and the bound ferredoxin was measured (Fig. 1).By this method, the stoichiometry of the CODHferredoxin complex wascalculated to be 1:l.The dependence of ionic strength on the complex formation was tested (Fig. 2). It has been observed that at an ionic strength of 1 M, where an electrostatically stabilized complexwouldbe expected to dissociate, the same amount of ferredoxin was detected in the filtrate tihne presence as well as inthe absence
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