Cytochrome b 5 (cyt b 5 ) is a 15-kDa amphipathic protein with a cytosolic amino-terminal catalytic heme domain, which is anchored to the microsomal membrane by a hydrophobic transmembrane ␣-helix at its carboxyl terminus. These two domains are connected by an ϳ15-amino acid linker domain, Ser 90 -Asp 104 , which has been modified by site-directed mutagenesis to investigate whether the length or sequence of the linker influences the ability of cyt b 5 to bind ferric cytochrome P450 2B4 and donate an electron to oxyferrous (cyt P450 2B4), thereby stimulating catalysis. Because shortening the linker by 8 or more amino acids markedly inhibited the ability of cyt b 5 to bind cyt P450 2B4 and stimulate catalysis by this isozyme, it is postulated 7 amino acids are sufficient to allow a productive interaction. All mutant cyts b 5 except the protein lacking the entire 15-amino acid linker inserted normally into the microsomal membrane. Alternatively, lengthening the linker by 16 amino acids, reversing the sequence of the amino acids in the linker, and mutating conserved linker residues did not significantly alter the ability of cyt b 5 to interact with cyt P450 2B4. A model for the membranebound cyt b 5 -cyt P450 complex is presented.Microsomal cytochrome b 5 (cyt b 5 ) 1 is an amphipathic electron transfer hemoprotein located in the membrane of the endoplasmic reticulum. It provides electrons for a broad range of reactions, including fatty acid desaturation, cholesterol biosynthesis, and a variety of cytochrome P450-dependent oxidation and hydroxylation reactions (1, 2).Cytochrome P450 (cyt P450) is responsible for the oxidation of a large number of substrates. The overall stoichiometry of the reaction catalyzed by cyt P450 is shown in Reaction 1, where RH is the substrate (3).The cyt P450 catalytic reaction occurs via a reaction cycle that involves substrate binding, reduction of the ferric heme, O 2 binding, reduction of the oxyferrous heme (second electron transfer), substrate oxidation, and finally product dissociation (4). Both ferric and oxyferrous cyt P450 receive electrons from NADPH via its redox partner, NADPH-cytochrome P450 reductase (cyt P450 reductase), whereas cyt b 5 can donate the second electron. Because of the high redox potential of Х20 mV for cyt b 5 , the first electron required to reduce cyt P450 from the ferric to the ferrous state is always transferred from NADPH via cyt P450 reductase. However, as the redox potential of cyt P450 increases from ХϪ230 mV to ϩ50 mV on transition to the oxyferrous state, the second electron can be obtained from either cyt P450 reductase or cyt b 5 (5, 6).Depending on the substrate, isozyme of cyt P450 and the experimental conditions cyt b 5 can stimulate, inhibit, or have no effect on cyt P450 activity (2). Studies of the stoichiometry of the metabolism of benzphetamine and methoxyflurane by cyt P450 2B4 have shown that cyt b 5 increases the efficiency of catalysis by cyt P450 2B4 primarily by decreasing the formation of the side-product superoxide. However, cyt b...