THE CO STRETCHING MODE INFRARED-SPECTRUM OF SUBSTRATE-FREE CYTOCHROMEP-450(CAM)-CO - THE EFFECT OF SOLVENT CONDITIONS, TEMPERATURE, AND PRESSURE

The effect of pH, glycerol, temperature, and pressure on the carbon mo noxide (GO) stretch mode of substrate-free cytochrome P-450(cam) (CYP1 01) was studied. Complex spectra of overlapping bands have been observ ed. CO stretch bands centered at about 1911-1918 cm(-1) (band I), 1927 -1931 cm(-1) (band II), 1940-1942 cm(-1) (band III), 1950-1953 cm(-1) (band IV), 1960-1963 cm(-1) (band V) and 1966-1973 cm(-1) (band VI) ar e obtained from the fitting analyses independently of the lineshape mo del used. Only two or three bands are dominant in each spectrum. Compa red to bands I, II and III, the bands IV and V are assigned to corresp ond 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 inte rpreted as indicating conformational substates of the protein. It is s uggested that water in the heme pocket plays an important role for the substate equilibrium. This substate equilibrium freezes in at the gla ss transition temperature: T-g, of the protein/solvent mixture. For th e temperature region above T, the thermodynamic parameters and volumes for the substates have been determined by a global fit analysis of th e temperature- and pressure-dependent populations and they are compare d to the respective values for camphor-bound cytochrome P-450(cam).