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 vFe-C = 530 cm-1 and .delta.FeCO = 585 cm-1, and form I, with vFe-C = 495 cm-1 and no detectable .delta.FeCO. They appear to have coincident vCO 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 vFeC = 503 cm-1, .delta.FeC = 575 cm-1, and vCO=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. Aged protein showed a substantial low-spin FeIII component and a large fraction of form I, even at elevated CO concentration. In addition, the phosphate inhibition of the form II .fwdarw. form II'' conversion is lost upon aging. A model for CO binding is put forward, which involves alternative conformations of the distal residues. The equilibrium is influenced by the binding of protons, phosphate, and a second CO molecule and is sensitive to irreversible protein changes that occur upon aging.