title: Raman and infrared spectra of cytochrome c peroxidase-carbon monoxide adducts in alternative conformational states.
abstract: Resonance Raman (RR) spectra are reported for CO-bound cytochrome c peroxidase (CCP). At low pH, two forms are observed: form II, with nu Fe-C = 530 cm-1 and delta FeCO = 585 cm-1, and form I, with nu Fe-C = 495 cm-1 and no detectable delta FeCO. They appear to have coincident nu CO infrared bands, at 1922 cm-1. These low-pH forms, similar to those observed for horseradish peroxidase (HRP), are attributed to tilted, H-bonded CO and perpendicular CO, respectively. The frequencies differ between the two proteins, a weaker H bond to CO being indicated for CCP. As with HRP, the equilibrium between forms I and II is shifted toward the latter at increasing CO concentrations, suggesting that secondary binding of CO perturbs the distal residues. At high pH [8.4, tris(hydroxymethyl)aminomethane buffer] the form II fraction converts to another form, II', with nu FeC = 503 cm-1, delta FeCO = 575 cm-1, and nu CO = 1948 cm-1; a tilted, non-H-bonded geometry is suggested. If phosphate buffer is used, however, form II (H bonded) persists at pH 8.4. This result establishes a role for phosphate in stabilizing the H-bonded form of the enzyme; it is suggested that phosphate binds near the distal imidazole and substantially increases its pKa. The conformational state is also influenced by aging. Fresh protein contains purely high spin FeIII heme, as monitored by the high-frequency RR spectrum, and yields form II almost exclusively at elevated CO concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)
mesh_terms: D002248:Carbon Monoxide / Q000378:metabolism*; D003578:Cytochrome-c Peroxidase / Q000378:metabolism*; D007700:Kinetics; D010544:Peroxidases / Q000378:metabolism*; D011487:Protein Conformation; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D013055:Spectrophotometry, Infrared / Q000379:methods; D013059:Spectrum Analysis, Raman / Q000379:methods
chemical_list: D002248:Carbon Monoxide; D010544:Peroxidases; D003578:Cytochrome-c Peroxidase