Characterization of two cysteine residues in cytochrome P-450scc: chemical identification of the heme-binding cysteine residue.
It was found that there were only two cysteine residues in highly purified cytochrome P-450scc molecule from bovine adrenocortical mitochondria by titration with 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) in denatured conditions. Only one cysteine residue at position 303 of cytochrome P-450scc could be specifically modified with DTNB in the native state. The resulting cytochrome P-450scc-5-thio-2-nitrobenzoic acid complex (cytochrome P-450scc-TNB) showed no distinct differences in absorption spectra, cholesterol binding, or electron transferring from adrenodoxin, compared to those of untreated cytochrome P-450scc. These observations indicated that the 303rd cysteine residue does not play a role in heme binding, cholesterol (substrate) binding or adrenodoxin binding. The other cysteine residue at 461 could be modified with DTNB only in a denatured condition. These assignments of cysteine residues were made by the subsequent S-cyanylation with KCN followed by incubation in 6 M guanidine hydrochloride at alkaline pH, which causes enhanced cleavage of peptide bonds adjacent to the cyanylated cysteine residues. Analyses of fragmented polypeptides by SDS-polyacrylamide gel electrophoresis confirmed that there were only two cysteine residues in the molecule and indicated that the cleavage rate of the peptide bond between 460 and 461 becomes high only when both cysteine residues (303 and 461) are cyanylated. These results clearly established that the 461st cysteine residue in cytochrome P-450scc plays a role as the heme fifth ligand on the basis of the general agreement that a thiolated cysteine residue coordinates to the heme iron.
M
M
Tsubaki
S
S
Tomita
Y
Y
Tsuneoka
Y
Y
Ichikawa
D000302:Adrenal Cortex / Q000201:enzymology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003545:Cysteine / Q000032:analysis*; D003577:Cytochrome P-450 Enzyme System / Q000032:analysis* / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000378:metabolism*; D007527:Isoenzymes / Q000032:analysis* / Q000378:metabolism; D007700:Kinetics; D008433:Mathematics; D008970:Molecular Weight; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; D004228:Dithionitrobenzoic Acid; D003545:Cysteine