Replacement of the Axial Histidine Ligand with Imidazole in Cytochrome c Peroxidase. 2. Effects on Heme Coordination and Function

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• 作者：Judy Hirst ; Sheri K. Wilcox ; Jingyuan Ai ; Pierre Moë ; nne-Loccoz ; Thomas M. Loehr ; and David B. Goodin
• 刊名：Biochemistry
• 出版年：2001
• 出版时间：February 6, 2001
• 年：2001
• 卷：40
• 期：5
• 页码：1274 - 1283
• 全文大小：106K
• 年卷期：v.40,no.5(February 6, 2001)
• ISSN：1520-4995

文摘

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 understandingof the role of axial ligands in heme catalysis. Here we report the functional and spectroscopic propertiesof 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 andtheir dependence on buffer and pH. UV-vis, EPR, and resonance Raman spectra reveal multiplecoordination states for H175G/Im which can be correlated with the crystal structures to assign the followingheme environments: H175G/H₂O/H₂O, H175G/Im_d/phosphate_c, H175G/Im_d/H₂O_c, H175G/Im_c/H₂O_d, andH175G/Im_c/OH^-_c, where H175G/X/Y defines the proximal species as X and the distal species as Y andc and d subscripts refer, where known, to the coordinated and dissociated states, respectively. ResonanceRaman data for reduced H175G/Im show two substates for heme-coordinated Im differing in the strengthof their hydrogen bond to Asp-235, in a fashion similar to WT CCP. NO binding to ferrous H175G/Imresults in dissociation of Im from the heme but not from the cavity, while no dissociation is observed forWT 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 dramaticallydifferent anisotropy and spin relaxation from that of the Trp-191 radical of WT CCP. It is suggested thatthese signals arise from alternate conformations of Trp191 having different degrees of exchange couplingto the ferryl heme, possibly mediated by the conformational heterogeneity of Im within the H175G cavity.The kinetics of the reaction of H175G/Im with H₂O₂ are multiphasic, also reflecting the multiplecoordination states of Im. The rate of the fastest phase is essentially identical to that of WT CCP, indicatingthat the H175G/Im_c/H₂O_d state is fully reactive with peroxide. However, the overall rate of enzyme turnoverusing cytochrome c as a substrate is <5% of WT and is unaffected by Im coordination. In summary, Imcoordination to H175G results in a number of conformers, one of which is structurally and spectroscopicallyvery similar to WT CCP. However, while this form is fully reactive with peroxide, the reaction withcytochrome c remains inefficient, perhaps implicating the altered Trp-191 radical species.