Kinetics of the reduction of cytochrome b5 with mutations in its membrane-binding domain

Abstract

In an attempt to understand which amino acids in the membrane anchor of cytochrome b5 might be determinants of its ability to support the cytochrome P450-catalyzed oxidation of selected substrates, the synthetic rat cytochrome b5 gene has been mutated by site-directed mutagenesis. The mutant proteins have been expressed in Saccharomyces cerevisiae, purified and assayed for their ability to support the cytochrome P450-catalyzed metabolism of the cytochrome b5 requiring substrate methoxyflurane (G. Vergères and L. Waskell, 1992, J. Biol. Chem. 267, 12583-12591). The rate of reduction of the cytochromes b5 by cytochrome P450 reductase has been examined by stopped-flow spectrophotometry to determine whether an altered rate of reduction of cytochrome b5 could explain the observed activity of cytochrome b5 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 system 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 b5 in 0.025% Tergitol and 40 microM dilauroylphosphatidylcholine suggests that this slow rate of reduction may be explained partially by aggregation of the polyleucine protein. The Pro115Stop mutant protein, which has been truncated by 19 amino acids in its COOH terminus resulting in a protein with one-half of the putative membrane anchor, supports methoxyflurane oxidation at 12-20% of the rate of the wild type protein. In addition it is reduced by cytochrome P450 reductase at a rate which should be capable of supporting a normal rate of production formation. The fact that the Pro115Stop mutant can be reduced at a rate capable of supporting a normal rate of methoxyflurane oxidation but in fact only supports methoxyflurane oxidation at 30% of the normal rate suggests that the mutant protein is deficient in its interactions with cytochrome P450. The mutant proteins, Pro115Ala and Ala116Pro, behaved essentially as did the wild type protein demonstrating that the presence (Pro115Ala) or absence (Ala116Pro) of an alpha helix in the middle of the putative membrane-binding domain of cytochrome b5 was not a determinant of the interaction of cytochrome b5 with cytochrome P450 reductase and cytochrome P450.(ABSTRACT TRUNCATED AT 400 WORDS)