Replacement of the Axial Histidine Ligand with Imidazole in Cytochrome c Peroxidase. 2. Effects on Heme Coordination and Function ² Judy Hirst, ‡,§ Sheri K. Wilcox, ‡,| Jingyuan Ai, ⊥ Pierre Moe ¨nne-Loccoz, ⊥ Thomas M. Loehr, ⊥ and David B. Goodin* ,‡ Department of Molecular Biology, MB8, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, and Department of Biochemistry and Molecular Biology, Oregon Graduate Institute of Science and Technology, 20000 NW Walker Road, BeaVerton, Oregon 97006-8921 ReceiVed September 5, 2000; ReVised Manuscript ReceiVed NoVember 20, 2000 ABSTRACT: The inability of imidazole to complement function in the axial histidine deletion mutant, H175G, of yeast cytochrome c peroxidase has been an intriguing but unresolved issue that impacts our understanding of the role of axial ligands in heme catalysis. Here we report the functional and spectroscopic properties of H175G and of its complexes with imidazole. Combined with the crystal structures for these complexes, the data provide a detailed and consistent account of the modes of Im binding in the H175G cavity and their dependence on buffer and pH. UV-vis, EPR, and resonance Raman spectra reveal multiple coordination states for H175G/Im which can be correlated with the crystal structures to assign the following heme environments: H175G/H 2 O/H 2 O, H175G/Im d /phosphate c , H175G/Im d /H 2 O c , H175G/Im c /H 2 O d , and H175G/Im c /OH - c , where H175G/X/Y defines the proximal species as X and the distal species as Y and c and d subscripts refer, where known, to the coordinated and dissociated states, respectively. Resonance Raman data for reduced H175G/Im show two substates for heme-coordinated Im differing in the strength of their hydrogen bond to Asp-235, in a fashion similar to WT CCP. NO binding to ferrous H175G/Im results in dissociation of Im from the heme but not from the cavity, while no dissociation is observed for WT CCP, indicating that steric tethering may, in part, control NO-induced dissociation of trans ligands. H175G/Im forms an oxidized compound I state with two distinct radical species, each with a dramatically different anisotropy and spin relaxation from that of the Trp-191 radical of WT CCP. It is suggested that these signals arise from alternate conformations of Trp191 having different degrees of exchange coupling to the ferryl heme, possibly mediated by the conformational heterogeneity of Im within the H175G cavity. The kinetics of the reaction of H175G/Im with H 2 O 2 are multiphasic, also reflecting the multiple coordination states of Im. The rate of the fastest phase is essentially identical to that of WT CCP, indicating that the H175G/Im c /H 2 O d state is fully reactive with peroxide. However, the overall rate of enzyme turnover using cytochrome c as a substrate is <5% of WT and is unaffected by Im coordination. In summary, Im coordination to H175G results in a number of conformers, one of which is structurally and spectroscopically very similar to WT CCP. However, while this form is fully reactive with peroxide, the reaction with cytochrome c remains inefficient, perhaps implicating the altered Trp-191 radical species. Recent studies have shown that the axial histidine ligand in several heme proteins can be deleted and replaced by exogenous small ligands to generate a range of novel heme protein complexes (1-16). These results pose questions at a new level of detail about how heme coordination dictates function, and how the protein environment enforces or modulates the properties of the heme-ligand complex. Initial reports of the substitution of imidazole for histidine in myoglobin (Mb) 1 (1) and cytochrome c peroxidase (CCP) (2) provided crystallographic observations of imidazole coordination within the cavity formed by deletion of the native histidine. Spectroscopic data showed that in the absence of added ligands, the ferric state of both H93G Mb (14) and H175G CCP (9) exist as mixtures of water- and hydroxide-coordinated species. Subsequent studies have provided spectroscopic, kinetic, and thermodynamic param- eters for binding of various substituted imidazole (4-7, 15- 17) and thiolate (12, 18) ligands to the H93G mutant of ² This work was supported in part by grants from the National Institutes of Health (GM41049 and GM48495 to D.B.G, and GM34468 to T.M.L.) and by a Wellcome Trust Prize International Research Fellowship to J.H. * To whom correspondence should be addressed. Phone: (858) 784- 9892. Fax: (858) 784-2857. E-mail: dbg@scripps.edu. ‡ The Scripps Research Institute. § Current address: Medical Research Council, Dunn Human Nutri- tion Unit, Hills Rd., Cambridge, CB2 2XY, U.K. | Current address: Pharmacia Corporation, Department of Protein Science, 301 Henrietta St., Kalamazoo, MI 49001. ⊥ Oregon Graduate Institute of Science and Technology. 1 Abbreviations: CCP, cytochrome c peroxidase; CCP(MKT), cy- tochrome c peroxidase produced by expression in Escherichia coli containing Met-Lys-Thr at the N-terminus, Ile at position 53, and Gly at position 152; H175G, mutant in which His-175 is replaced by Gly; H175G/X c/d/Yc/d, state in which ligand X occupies the proximal heme cavity, ligand Y occupies the distal heme cavity, and these are either coordinated (c) to or dissociated (d) from the heme iron; WT, wild- type CCP; cyt c, cytochrome c; Mb, myoglobin; EPR, electron paramagnetic resonance; RR, resonance Raman; Im, imidazole. 1274 Biochemistry 2001, 40, 1274-1283 10.1021/bi002090q CCC: $20.00 © 2001 American Chemical Society Published on Web 01/11/2001