Cytochrome P450c17 catalyzes steroid 17alpha-hydroxylase and 17,20-lyase activities and hence is a key enzyme in the production of human glucocorticoids and sex steroids. These two activities are catalyzed in a single substrate-binding site but are regulated independently in human physiology. We have recently shown that cytochrome b5 facilitates 17,20-lyase activity by allosterically promoting the interaction of P450c17 with P450 oxidoreductase (OR) and that the human P450c17 mutations, R347H and R358Q, selectively destroy 17,20-lyase activity while sparing 17alpha-hydroxylase activity. We transfected COS-1 cells with vectors for these P450c17 mutants and found that an excess of OR and b5 restored a small amount of 17,20-lyase activity, suggesting the mutations interfere with electron donation. To determine whether these mutations selectively interfere with the interaction of P450c17 and its electron-donating system, we expressed each P450cl7 mutant in yeast with or without OR, b5, or both, and measured enzyme kinetics in yeast microsomes using pregnenolone and 17alpha-hydroxypregnenolone as substrates. The apparent Michaelis-Menten (Km) values for the R347H mutant with and without coexpressed OR were 0.2 and 0.6 microM, respectively, and for the R358Q mutant with and without OR they were 0.3 and 0.4 microM, respectively; these values did not differ significantly from the wild-type values of 0.4 and 0.8 microM with and without OR, respectively. Furthermore, coincubation with 17alpha-hydroxypregnenolone showed a competitive mechanism for interference of catalysis. The similar kinetics and the competitive inhibition prove that the mutations did not affect the active site. Coexpression of the mutants with OR yielded insignificant 17,20-lyase activity, but addition of a 30:1 molar excess cytochrome b5 to these microsomes restored partial 17,20-lyase activity, with the R358Q mutant achieving twice the activity of the R347H mutant. These data indicate that both mutations selectively interfere with 17,20-lyase activity by altering the interaction of P450c17 with OR, thus proving that the lyase activity was disrupted by interfering with electron transfer. Furthermore, the data offer the first evidence that R347 is a crucial component of the site at which b5 interacts with the P450c17 x OR complex to promote electron transfer.