The binding of
substrates to
heme enzymes has been widely assumed to occur at the so-called delta-heme edge. Recently, however, a number of examples have appeared in which
substrate binding at an alternative site, the gamma-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 gamma-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 gamma-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 pi-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 gamma-heme edge.
