Part I. Magnetic circular dichroism studies of cytochrome c peroxidase from yeast: Investigations of the active site heme coordination sphere through comparison with horseradish peroxidase and myoglobin. Part II. Examination of the heme active site coordination structure of the cavity mutants of myoglobin (H93G), cytochrome c peroxidase (H175G), and heme oxygenase (H25A) and structural investigation of thiolate-ligated cytochrome c peroxidase in the H17C/D235L double mutant.

摘要

Part I. Magnetic Circular Dichroism (MCD) and UV-Visible absorption spectroscopies have been used to study the effect of aging, pH and source on the active site heme coordination structure of exogenous ligand-free yeast cytochrome c peroxidase (CCP). Spectral analysis indicates that both wild type and recombinant forms of CCP are predominantly five-coordinate high spin at neutral pH. At alkaline pH (9.7), wild type CCP is a mixture of six-coordinate, predominantly low-spin complexes with the distal histidine (His 52), and hydroxide acting as distal ligands based on MCD spectral comparisons. Aged samples of CCP appear to contain considerably increased amounts of six-coordinate low-spin species including histidine/hydroxide and bis-histidine complexes. Additionally, various ferric, ferrous and ferryl complexes of CCP have been examined with MCD and compared to the analogous derivatives of horseradish peroxidase and myoglobin.;Part II. Spectral analysis of exogenous ligand-free ferric H93G Mb reveal that its heme coordination structure has a single water ligand at pH 5.0, a single hydroxide ligand at pH 10.0 and a mixture of species at pH 7.0 including five coordinate hydroxide, and various six-coordinate structures. Exogenous ligand-free ferric H25A heme oxygenase at neutral pH appears to be five coordinate with ligation by a carboxylate group of a nearby glutamic acid residue based on MCD comparison with model complexes. Examination of the MCD spectra for exogenous ligand-free ferric H175G CCP reveals the protein is coordinated by a single phosphate group at pH 5.9, and at pH 7.0 when in potassium phosphate buffer. Additionally, various mixed ligand complexes of imidazole-bound H93G Mb in the ferric, ferrous and ferryl oxidation states have been examined by MCD and routinely display spectral properties similar to wild type myoglobin.;Finally, the double mutant, H175C/D235L CCP, has been engineered and characterized by UV-Visible absorption, MCD and EPR spectroscopies at pH 8.0. Spectral analysis reveals that the double mutant is five-coordinate, high-spin with thiolate ligation in the exogenous ligand-free ferric state. Addition of imidazole or cyanide results in the formation of a six-coordinate low-spin complex which retains thiolate ligation. All three complexes are comparable to the analogous forms of cytochrome P450cam.