The hypothesis is examined that during mixed-function oxidation reactions catalyzed by hepatic microsomes, cytochrome b₅ may function by transferring reducing equivalents originating from either DPNH or TPNH to cytochrome P-450, thereby providing one of the two electrons required for cytochrome P-450 function. The conclusion that cytochrome b₅ participates in this reaction is based on the observation of a partial reoxidation of reduced cytochrome b₅ in the presence of excess DPNH and substrate, when mixed-function oxidation reactions are initiated by the addition of TPNH. The steady-state change of reduced cytochrome b₅ is dependent on the presence of oxygen as well as substrate and coincides with product formation and oxygen utilization.

No evidence was obtained for a direct interaction of DPNH with cytochrome P-450 in support of TPNH-dependent hydroxylation reactions. DPNH neither supports TPNH-dependent lipid peroxidation nor has it any stimulating effect upon the anaerobic reduction of cytochrome P-450 in the presence of saturating concentrations of TPNH. Furthermore, it is concluded that the DPNH enhancement of the overall hydroxylation reaction is dependent on the presence of an apparent rate-limiting step in the TPNH-dependent reaction as demonstrated by modifying this step with cations. These observations suggest that cytochrome b₅ interacts with a form of cytochrome P-450 in a concerted mechanism at a step after the primary reduction of cytochrome P-450 by reducing equivalents originating from TPNH. The implications of these findings on the apparent rate-limiting step for the overall hydroxylation reaction is discussed.