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

The inability of imidazole to complement function in the axial histidine de letion mutant, H175G, of yeast cytochrome c peroxidase has been an intrigui ng 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 co nsistent account of the modes of Im binding in the H175G cavity and their d ependence on buffer and pH. W-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/H2O/H2O , H175G/Im(d)/phosphate,, H175G/Im(d)/H2Oc, H175G/Im(c)/H2Od, and H175G/Im( c)/OHc-, where H175G/X/Y defines the proximal species as X and the distal s pecies as Y and c and d subscripts refer, where known, to the coordinated a nd 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 K175G/Im results in dissociation of Im from the heme but not fr om the cavity, while no dissociation is observed for WT CCP, indicating tha t steric tethering may, in part, control NO-induced dissociation of trans l igands. H175G/Im forms an oxidized compound I state with two distinct radic al species, each with a dramatically different anisotropy and spin relaxati on from that of the Trp-191 radical of WT CCP. It is suggested that these s ignals arise from alternate conformations of Trp191 having different degree s of exchange coupling to the ferryl heme, possibly mediated by the conform ational heterogeneity of Im within the H175G cavity. The kinetics of the re action of H175G/Im with H2O2 are multiphasic, also reflecting the multiple coordination states of Im. The rate of the fastest phase is essentially ide ntical to that of WT CCP, indicating that the H175G/Im(c)/H2Od state is ful ly reactive with peroxide. However, the overall rate of enzyme turnover usi ng cytochrome c as a substrate is <5% of WT and is unaffected by Im coordin ation. In summary, Im coordination to H175G results in a number of conforme rs, one of which is structurally and spectroscopically very similar to WT C CP. However, while this form is fully reactive with peroxide, the reaction with cytochrome c remains inefficient, perhaps implicating the altered Trp- 191 radical species.