KINETICS OF THE REDUCTION OF CYTOCHROME B(5) WITH MUTATIONS IN ITS MEMBRANE-BINDING DOMAIN

In an attempt to understand which amino acids in the membrane anchor o f cytochrome b(5) might be determinants of its ability to support the cytochrome P450-catalyzed oxidation of selected substrates, the synthe tic rat cytochrome b(5) gene has been mutated by site-directed mutagen esis. The mutant proteins have been expressed in Saccharomyces cerevis iae, purified and assayed for their ability-to support the cytochrome P450-catalyzed metabolism of the cytochrome b(5) requiring substrate m ethoxyflurane (G. Vergeres and L. Waskell, 1992, J. Biol. Chem. 267, 1 2583-12591). The rate of reduction of the cytochromes b(5) by cytochro me P450 reductase has been examined by stopped-flow spectrophotometry to determine whether an altered rate of reduction of cytochrome b(5) c ould explain the observed activity of cytochrome b(5), in the purified reconstituted mixed-function oxidase system. A mutant in which the 22 -amino-acid membrane anchor was replaced by a sequence of 22 leucines was unable to support methoxyflurane metabolism in the reconstituted s ystem and was reduced by cytochrome P450 reductase at a rate (k = 4.5 X 10(-3) s(-1)) slow enough to explain this finding. Comparison of the rate of reduction of this mutant cytochrome b(5) in 0.025% Tergitol a nd 40 mu M dilauroylphosphatidylcholine suggests that this slow rate o f reduction may be explained partially by aggregation of the polyleuci ne protein. The Pro115Stop mutant protein, which has been truncated by 19 amino acids in its COOH terminus resulting in a protein with one-h alf of the putative membrane anchor, supports methoxyflurane oxidation at 12-20% of the rate of the wild type protein. In addition it is red uced by cytochrome P450 reductase at a rate which should be capable of supporting a normal rate of product formation. The fact that the Pro1 15Stop mutant,can be reduced at a rate capable of supporting a normal rate of methoxyflurane oxidation but in fact only supports methoxyflur ane oxidation at 30% of the normal rate suggests that the mutant prote in is deficient in its interactions with cytochrome P450. The mutant p roteins, Pro115Ala and Ala116Pro, behaved essentially as did the wild type protein demonstrating that the presence (Pro115Ala) or absence (A la116Pro) of an alpha helix in the middle of the putative membrane-bin ding domain of cytochrome b(5) was not a determinant of the interactio n of cytochrome b(5) with cytochrome P450 reductase and cytochrome P45 0. The double mutant Ala131Lys; Glu132Lys in which the net negative ch arge at the C-terminus was altered to a net positive charge also funct ioned in a manner identical to that of the wild type protein, indicati ng that the net charge at the carboxyl terminus was not essential to t he interaction of cytochrome b(5) with its electron transfer partners. (C) 1994 Academic Press, Inc.