The CO stretching mode infrared spectrum of substrate-free cytochrome P-450_cam-CO: The effect of solvent conditions, temperature, and pressure

The effect of pH, glycerol, temperature, and pressure on the carbon monoxide (CO) stretch mode of substrate-free cytochrome P-450_cam (CYP101) was studied. Complex spectra of overlapping bands have been observed. CO stretch bands centered at about 1911-1918 cm¹ (band I), 1927-1931 cm¹ (band II), 1940-1942 cm¹ (band III), 1950-1953 cm¹ (band IV), 1960-1963 cm¹ (band V) and 1966-1973 cm¹ (band VI) are obtained from the fitting analyses independently of the lineshape model used. Only two or three bands are dominant in each spectrum. Compared to bands I, II and III, the bands IV and V are assigned to correspond to a weaker polar contact between the CO ligand and a polar group in the heme pocket (probably Thr252) because of the opposite effect of glycerol (osmotic pressure) and hydrostatic pressure on the intensity and frequency of these bands. The different CO stretch bands are interpreted as indicating conformational substates of the protein. It is suggested that water in the heme pocket plays an important role for the substate equilibrium. This substate equilibrium freezes in at the glass transition temperature, T_g, of the protein/solvent mixture. For the temperature region above T_g the thermodynamic parameters and volumes for the substates have been determined by a global fit analysis of the temperature- and pressure-dependent populations and they are compared to the respective values for camphor-bound cytochrome P-450_cam.