Interaction of Catalytic Domains in Cytochrome P450scc-Adrenodoxin Reductase-Adrenodoxin Fusion Protein Imported into Yeast Mitochondria

L. A. Novikova¹, P. A. Nazarov¹, A. S. Saveliev¹, V. L. Drutsa¹,V. N. Sergeev¹, W. L. Miller², and V. N. Luzikov^1*

¹Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (095) 939-3181; E-mail: luzikov@biogen.genebee.msu.su

²Department of Pediatrics and The Metabolic Research Unit, University of California, San Francisco, 94143-0978 USA

^* To whom correspondence should be addressed.

Received March 13, 2000; Revision received March 28, 2000

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We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc-adrenodoxin reductase-adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted.

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KEY WORDS: cytochrome P450scc, adrenodoxin, NADPH:adrenodoxin reductase, fusion protein, enzymatic activity, yeast, mitochondria, proteolysis

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