ALTERING SUBSTRATE-SPECIFICITY AT THE HEME EDGE OF CYTOCHROME-C PEROXIDASE

Two mutants of cytochrome c peroxidase (CCP) are reported which exhibi t unique specificities toward oxidation of small substrates. Ala-147 i n CCP is located near the delta-meso edge of the heme and along the so lvent access channel through which H2O2 is thought to approach the act ive site, This residue was replaced with Met and Tyr to investigate th e hypothesis that small molecule substrates are oxidized at the expose d delta-meso edge of the heme. X-ray crystallographic analyses confirm that the side chains of A147M and A147Y are positioned over the delta -meso heme position and might therefore modify small molecule access t o the oxidized heme cofactor. Steady-state kinetic measurements show t hat cytochrome c oxidation is enhanced 3-fold for A147Y relative to wi ld type, while small molecule oxidation is altered to varying degrees depending on the substrate and mutant. For example, oxidation of pheno ls by A147Y is reduced to less than 20% relative to the wild-type enzy me, while V-max/e for oxidation of other small molecules is less affec ted by either mutation. However, the ''specificity'' of aniline oxidat ion by A147M, i.e., (V-max/e)/K-m, is 43-fold higher than in wild-type enzyme, suggesting that a specific interaction for aniline has been i ntroduced by the mutation. Stopped-flow kinetic data show that the res tricted heme access in A147Y or A147M slows the reaction between the e nzyme and H2O2, but not to an extent that it becomes rate limiting for the oxidation of-the substrates examined. The rate constant for compo und ES formation with A147Y is 2.5 times slower than with wild-type CC P. These observations strongly support the suggestion that small molec ule oxidations occur at sites on the enzyme distinct from those utiliz ed by cytochrome c and that the specificity of small molecule oxidatio n can be significantly modulated by manipulating access to the heme ed ge. These results help to define the role of alternative electron tran sfer pathways in cytochrome c peroxidase and may have useful applicati ons in improving the specificity of peroxidases with engineered functi on.