title: Cytochrome c peroxidase mutant active site structures probed by resonance Raman and infrared signatures of the CO adducts.
abstract: Vibrational frequencies associated with FeC and CO stretching and FeCO bending modes have been determined via resonance Raman (RR) and infrared (IR) spectroscopy for cytochrome c peroxidase (CCP) mutants prepared by site-directed mutagenesis. These include the bacterial "wild type", CCP(MI), and mutations involving groups on the proximal (Asp-235----Asn; Trp-191---Phe) and distal (Trp-51----Phe; Arg-48----Leu and Lys) side of the heme. The data were analyzed with the aid of a recently established correlation between nu FeC and nu CO, which can be used to distinguish between back-bonding and axial ligand donor effects. At high pH all adducts showed essentially the same vibrational pattern (form I') with nu FeC approximately 505 cm-1, nu CO approximately 1948 cm-1, and delta FeCO (weak RR band) approximately 576 cm-1. These frequencies are very similar to those shown by the myoglobin CO adduct and imply a "normal" H-bond of the proximal histidine. At pH 7 (pH 6 for Asn-235 and Leu-48), different forms are seen for different proteins: form I (nu FeC approximately 500 cm-1, nu CO = 1922-1941 cm-1, and delta FeCO approximately 580 cm-1, very weak) in the case of CCP(MI) and Phe-191, as well as bakers' yeast CCP, or form II (nu FeC approximately 530 cm-1, nu CO = 1922-1933 cm-1, and delta FeCO = 585 cm-1, moderately strong) for Asn-235 and Phe-51.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Dipartimento di Chimica, Università di Firenze, Italy.
authors.forename: G
authors.initials: G
authors.lastname: Smulevich
authors.forename: J M
authors.initials: JM
authors.lastname: Mauro
authors.forename: L A
authors.initials: LA
authors.lastname: Fishel
authors.forename: A M
authors.initials: AM
authors.lastname: English
authors.forename: J
authors.initials: J
authors.lastname: Kraut
authors.forename: T G
authors.initials: TG
authors.lastname: Spiro
mesh_terms: D001120:Arginine; D001665:Binding Sites; D002248:Carbon Monoxide; D003578:Cytochrome-c Peroxidase; D004252:DNA Mutational Analysis; D006418:Heme; D006639:Histidine; D006860:Hydrogen Bonding; D010544:Peroxidases; D011487:Protein Conformation; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013055:Spectrophotometry, Infrared; D013059:Spectrum Analysis, Raman
chemical_list: D011994:Recombinant Proteins; D006418:Heme; D006639:Histidine; D002248:Carbon Monoxide; D001120:Arginine; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase