Enzymatic properties of vesicle-reconstituted human cytochrome P450SCC (CYP11A1) - Differences in functioning of the mitochondrial electron-transfer chain using human and bovine adrenodoxin and activation by cardiolipin

The recently reported heterologous expression and purification of both huma n cytochrome P450SCC and adrenodoxin [Woods, SIT., Sadleir, J., Downs, T., Triantopoulos, T., Haedlam, M.J. & Tuckey, R.C. (1998) Arch. Biochem. Bioph ys. 353, 109-115] has enabled us to perform studies with the membrane-recon stituted human enzymes to better understand the side-chain cleavage reactio n in humans. Human P450SCC was successfully reconstituted into dioleoylphos phatidylcholine vesicles with and without cardiolipin and its enzymatic pro perties characterized in the membrane-bound state. Enhancement of the P450S CC activity and significant activation by cardiolipin were observed when hu man adrenodoxin instead of bovine adrenodoxin was used as electron donor. I n the absence of cardiolipin, K-m for cholesterol was decreased twice in th e case of human adrenodoxin indicating enhanced cholesterol binding. On the other hand, in the presence of cardiolipin in the membrane both K-m and V for cholesterol were decreased with human adrenodoxin as electron donor. Ki netic analysis of the interaction between human P450SCC and its redox partn ers provided evidence for enhanced binding of the human electron donor to h uman P450SCC indicated by both an increased V and decreased K-d for human a drenodoxin compared with the values with bovine adrenodoxin. Because no sim ilar effects were observed in Tween 20 micelles, these results suggest that the phospholipid membrane may play an important role in the interaction of human adrenodoxin with human P450SCC.