Effects of temperature and glycerol on the resonance Raman spectra of cytochrome c peroxidase and selected mutants.
The high-frequency resonance Raman spectra of FeIII yeast native cytochrome c peroxidase (CCP) and five of its mutants [CCP(MI), Phe-51, Leu-48, Lys-48, Asn-235, and Phe-191] were recorded in phosphate buffer, pH 7.0, and in glycerol/phosphate mixtures at 295 and 10 K. Glycerol induces heme coordination changes in some of the CCP mutants at room temperature. It apparently weakens the binding of the Fe atom to ligands in the distal heme cavity and drives the heme toward the 5-coordinate, high-spin state. At 10 K, native CCP and all the mutants (except Phe-51 which remains 6-coordinate, high-spin) show various distributions of spin and coordination states which differ from those observed at 295 K. Upon cooling in phosphate buffer, pH 7, and to a much lesser extent in 66% glycerol/phosphate, an internal strong-field ligand is coordinated to the Fe. A likely candidate is H2O-595, which could become a strong-field ligand on H-bonding and/or proton transfer to H2O-648, and/or the distal His-52. However, distal His-52 itself cannot be ruled out as the coordinating ligand considering that the Phe-51 mutant, which binds H2O-595 at room temperature, does not show a large 6-coordinate, low-spin component at 10 K like the other mutants. These results clearly indicate that the Fe coordination in CCP and its mutants is sensitive to both temperature and solvent composition.
Smulevich
G
G
Dipartimento di Chimica, Universitá di Firenze, Italy.
Mantini
A R
AR
English
A M
AM
Mauro
J M
JM
D003578:Cytochrome-c Peroxidase / Q000032:analysis* / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000378:metabolism; D005822:Genetic Vectors; D005990:Glycerol; D006418:Heme / Q000032:analysis; D009154:Mutation; D010544:Peroxidases / Q000032:analysis*; D010957:Plasmids; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013059:Spectrum Analysis, Raman; D013696:Temperature
D006418:Heme; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase; D005990:Glycerol