Engineering the Substrate Specificity and Reactivity of a Heme Protein: Creation of an Ascorbate Binding Site in Cytochrome c Peroxidase † Emma J. Murphy, ‡ Clive L. Metcalfe, ‡ Jaswir Basran, § Peter C. E. Moody, § and Emma Lloyd Raven ‡, * Department of Chemistry, Henry Wellcome Building, UniVersity of Leicester, UniVersity Road, Leicester, LE1 9HN, England U.K., and Department of Biochemistry and Henry Wellcome Laboratories for Structural Biology, Henry Wellcome Building, UniVersity of Leicester, Lancaster Road, Leicester, LE1 9HN, England U.K. ReceiVed August 6, 2008; ReVised Manuscript ReceiVed NoVember 7, 2008 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. Our understanding of substrate binding across various heme enzymes developed largely from crystallographic information for a number of heme peroxidase enzymes. The first structures to appear showed binding of aromatic substrates close to the so-called δ-heme edge (1-5), and these structures were consistent with other observations, for example from earlier chemical modification work (6-8), in which substrate binding at the δ-heme edge was also implicated. As a consequence, a consensus emerged in which substrate binding and oxidation at the so-called δ-heme edge was widely assumed. With the exception of the cytochrome c peroxidase/cytochrome c complexswhich was known to be anomalous in part because of its unusual substratesthe only outlier to this general “trend” was the structure for the manganese peroxidase/Mn(II) complex, which showed Mn(II) bound at a different location, close to the γ-heme edge and ligated by carboxylate groups and the heme 6-propionate (9). Later on, two other structures appeared, for the ascorbate peroxidase/ascorbate (10) and nitric oxide synthase/ tetrahydrobiopterin (11, 12) complexes. These structures also revealed hydrogen bonding interactions between the substrate and the heme 6-propionate at the γ-heme edge. It became clear, therefore, that substrate binding at the δ-heme edge was not the only means by which the enzyme and substrate might productively associate with one another. As far as the protein is concerned, this offers distinct advantages over the “one-site-fits-all” model, because it provides more than one route through which electron delivery can be channeled. Consequently, oxidation of different types of substrate can be accommodated within the same protein framework, for example in ascorbate peroxidase where (hydrophobic) aromatic substrates and (hydrophilic) ascorbate are oxidized at different sites (1, 10). In previous work (10), we identified the ascorbate binding site in ascorbate peroxidase (APX 1 ), Figure 1. The structure was helpful not only because it revealed the details of the binding interactions in APX but also because but also because it helped to rationalize differences with the closely related cytochrome c peroxidase enzyme. Hence, we noted that the residues required for ascorbate binding (Arg172, Lys 30, Figure 1 (inset)) are replaced by Asn184 and Asp33 in CcP; conversely, the residues required for binding of cytochrome c (Asp34, Glu35, Asp37, Glu290 (13)) are completely missing in APX. This comparison of the two structures also accounted for the observation that Trp179 is not essential for catalysis in APX (14) while the equivalent residue (Trp191) in CcP is (15), because in APX there is direct coupling of the substrate to the heme propionate, completely bypassing Trp179. In this work, we demonstrate that CcP can, with relatively modest changes in protein structure † This work was supported by grants from the BBSRC (Project Grants BB/C00602X/1, BB/C001184/1 and IIP 0206 /009, and a studentship to E.J.M.). * To whom correspondence should be addressed. Tel: +44 (0)116 2297047. Fax: +44 (0)116 252 2789. E-mail: emma.raven@le.ac.uk. ‡ Department of Chemistry. § Department of Biochemistry and Henry Wellcome Laboratories for Structural Biology. 1 Abbreviations: APX, ascorbate peroxidase; CcP, cytochrome c peroxidase. Biochemistry 2008, 47, 13933–13941 13933 10.1021/bi801480r CCC: $40.75  2008 American Chemical Society Published on Web 12/05/2008