Engineering the Substrate Specificity and Reactivity of a Heme Protein: Creation of an Ascorbate Binding Site in Cytochrome c Peroxidase

Abstract

The binding of substrates to heme enzymes has been widely assumed to occur at the so-called δ-heme edge. Recently, however, a number of examples have appeared in which substrate binding at an alternative site, the γ-heme edge, is also possible. In previous work [Sharp et al. (2003) Nat. Struct. Biol.10, 303−307], we showed that binding of ascorbate to ascorbate peroxidase occurred at the γ-heme edge. Here, we show that the closely related cytochrome c peroxidase enzyme can duplicate the substrate binding properties of ascorbate peroxidase through the introduction of relatively modest structural changes at Tyr36 and Asn184. Hence, crystallographic data for the Y36A/N184R/W191F triple variant of cytochrome c peroxidase shows ascorbate bound to the γ-heme edge, with hydrogen bonds to the heme propionate and Arg184. In parallel mechanistic studies in variants incorporating the W191F mutation, we show that a transient porphyrin π-cation radical in Compound I of cytochrome c peroxidase, analogous to that observed in ascorbate peroxidase, is competent for ascorbate oxidation but that under steady state conditions this intermediate decays too rapidly to sustain efficient turnover of ascorbate. The results are discussed in terms of our more general understanding of substrate oxidation across other heme proteins, and the emerging role of the heme propionates at the γ-heme edge.