Dissociation of bovine cytochrome c1 subcomplex and the status of cysteine residues in the subunits.
Purified bovine heart two-band cytochrome c1 subcomplex was dissociated by treatment with p-chloromercuribenzoic acid (pCMB) into its heme subunit and a colorless subunit called hinge protein, which is essential for the formation of cytochrome c1-c complex. The subcomplex was found by titration to react with 4 mol of pCMB per mol of cytochrome c1. The contents of mercury of the dissociated heme subunit and the hinge protein were 3 and 1 mol per mol of polypeptide, respectively. These results, together with the sequence analysis, indicated that the three cysteine residues in cytochrome c1 heme subunit not involved in heme-binding existed in free thiol form. One of the five cysteine residues in the hinge protein was in free form and four in two disulfide bonds. The dissociated hinge protein was digested with staphylococcal protease and the cysteine-containing peptides were separated by reversed-phase high-performance liquid chromatography (HPLC). The content of mercury and the result of performic acid oxidation of cystine peptides revealed that Cys-30 existed in free thiol form and two disulfide bridges were formed between Cys-24 and Cys-68 and between Cys-40 and Cys-54. The conformation of the hinge protein was predicted to be composed largely of either two-alpha-helical or four-alpha-helical conformation with the amino (N)-terminal 20 residues being in a random structure.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002729:Chloromercuribenzoates; D003545:Cysteine / Q000032:analysis*; D003574:Cytochrome c Group / Q000032:analysis*; D045304:Cytochromes c; D003575:Cytochromes c1; D004591:Electrophoresis, Polyacrylamide Gel; D005561:Formates; D006418:Heme / Q000032:analysis; D008628:Mercury / Q000032:analysis; D010084:Oxidation-Reduction; D011506:Proteins / Q000032:analysis; D013438:Sulfhydryl Compounds / Q000032:analysis
D000596:Amino Acids; D002729:Chloromercuribenzoates; D003574:Cytochrome c Group; D005561:Formates; D011506:Proteins; D013438:Sulfhydryl Compounds; C031949:cytochrome c(1)-cytochrome c complex; C008817:peroxyformic acid; D006418:Heme; D045304:Cytochromes c; D003575:Cytochromes c1; D008628:Mercury; D003545:Cysteine