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

J. Hirst, S. K. Wilcox, J. Ai, Pierre Moenne-Loccoz, T. M. Loehr, D. B. Goodin

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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/H2O/H2O, H175G/Imd/phosphatec, H175G/Imd/H2Oc, H175G/Imc/H2Od, and H175G/Imc/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 H2O2 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/Imc/H2Od state is fully reactive with peroxide. However, the overall rate of enzyme turnover using cytochrome c as a substrate is

Original languageEnglish (US)
Pages (from-to)1274-1283
Number of pages10
JournalBiochemistry
Volume40
Issue number5
DOIs
StatePublished - Feb 6 2001

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Cytochrome-c Peroxidase
Heme
Histidine
Ligands
Crystal structure
Exchange coupling
Anisotropy
Peroxides
Cytochromes c
Catalysis
Yeast
Paramagnetic resonance
Conformations
Raman scattering
Hydrogen
Hydrogen bonds
Buffers
Yeasts
imidazole
Kinetics

ASJC Scopus subject areas

  • Biochemistry

Cite this

Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 2. Effects on heme coordination and function. / Hirst, J.; Wilcox, S. K.; Ai, J.; Moenne-Loccoz, Pierre; Loehr, T. M.; Goodin, D. B.

In: Biochemistry, Vol. 40, No. 5, 06.02.2001, p. 1274-1283.

Research output: Contribution to journalArticle

Hirst, J. ; Wilcox, S. K. ; Ai, J. ; Moenne-Loccoz, Pierre ; Loehr, T. M. ; Goodin, D. B. / Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 2. Effects on heme coordination and function. In: Biochemistry. 2001 ; Vol. 40, No. 5. pp. 1274-1283.
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T1 - Replacement of the axial histidine ligand with imidazole in cytochrome c peroxidase. 2. Effects on heme coordination and function

AU - Hirst, J.

AU - Wilcox, S. K.

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AU - Loehr, T. M.

AU - Goodin, D. B.

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N2 - 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/H2O/H2O, H175G/Imd/phosphatec, H175G/Imd/H2Oc, H175G/Imc/H2Od, and H175G/Imc/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 H2O2 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/Imc/H2Od state is fully reactive with peroxide. However, the overall rate of enzyme turnover using cytochrome c as a substrate is

AB - 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/H2O/H2O, H175G/Imd/phosphatec, H175G/Imd/H2Oc, H175G/Imc/H2Od, and H175G/Imc/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 H2O2 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/Imc/H2Od state is fully reactive with peroxide. However, the overall rate of enzyme turnover using cytochrome c as a substrate is

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