Type I cytochrome P450 enzyme systems are found in mitochondria and consist of three components, a flavoprotein (adrenodoxin reductase, AdRed), an iron-sulfur protein (adrenodoxin, Adx), and the cytochrome P450; Type II P450 enzymes in the endoplasmic reticulum consist of only two components, P450 reductase and the P450. Genetically engineered fusion proteins of Type II cytochromes P450 (such as steroid 17 alpha- and 21-hydroxylases) produce enzymes with increased activity. To test the consequences of constructing fusions of Type I enzymes, we built fusion proteins based on the cholesterol side-chain cleavage enzyme, P450scc. We constructed expression vectors for three fusion proteins: NH2-P450scc-AdRed-COOH, P450-AdRed-Adx, and P450scc-Adx-AdRed. The various components were assembled from cassette-like cDNA fragments modified and amplified by polymerase chain reaction (PCR), subcloned into a specially tailored vector, and linked by DNA segments encoding hydrophilic linker peptides. The final vectors were transfected into COS-1 cells, incubated with 22R-hydroxycholesterol, and assayed by the secretion of pregnenolone into the culture medium. Triple transfection of three individual vectors expressing P450scc, AdRed, and Adx yielded more pregnenolone than did transfection with P450scc alone. The P450scc-AdRed and P450scc-Adx-AdRed fusion proteins produced levels of pregnenolone similar to the control triple transfection. However, the P450scc-AdRed-Adx fusion produced substantially more pregnenolone, having an apparent Vmax of 9.1 ng of pregnenolone produced per milliliter of medium per 24 hr, compared to a Vmax of 1.7 ng/ml per day for the triple transfection.(ABSTRACT TRUNCATED AT 250 WORDS)