Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 2. Effects on heme coordination and function.

TitleReplacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 2. Effects on heme coordination and function.
Publication TypeJournal Article
Year of Publication2001
AuthorsHirst, J, Wilcox, SK, Ai, J, Moënne-Loccoz, P, Loehr, TM, Goodin, DB
JournalBiochemistry
Volume40
Issue5
Pagination1274-83
Date Published2001 Feb 06
ISSN0006-2960
KeywordsAmino Acid Substitution, Binding Sites, Cytochrome-c Peroxidase, Electron Spin Resonance Spectroscopy, Escherichia coli, Ferrous Compounds, Free Radicals, Glycine, Heme, Histidine, Imidazoles, Ligands, Nitric Oxide, Protein Binding, Spectrophotometry, Ultraviolet, Spectrum Analysis, Raman
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.

DOI10.1021/bi002090q
Alternate JournalBiochemistry
Citation Key10.1021/bi002090q
PubMed ID11170453
Grant ListR01 GM041049 / GM / NIGMS NIH HHS / United States
GM34468 / GM / NIGMS NIH HHS / United States
GM41049 / GM / NIGMS NIH HHS / United States
GM48495 / GM / NIGMS NIH HHS / United States