Cytochrome c peroxidase mutant active site structures probed by resonance Raman and infrared signatures of the CO adducts.
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)
Smulevich
G
G
Dipartimento di Chimica, Università di Firenze, Italy.
Mauro
J M
JM
Fishel
L A
LA
English
A M
AM
Kraut
J
J
Spiro
T G
TG
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
D011994:Recombinant Proteins; D006418:Heme; D006639:Histidine; D002248:Carbon Monoxide; D001120:Arginine; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase