The effect of pH, glycerol, temperature, and pressure on the carbon inonoxide (CO) stretch mode of substrate-free cytochrome P-450,,,,, (CYP101) was studied. Complex spectra of overlapping bands have been observed. CO stretch bands centered at about 191 1-1918 cm ' (band I), 1927-1931 cm-' (band 11), 1940-1942cm-' (band 111), cm-' (band IV), 1960 -1963 cm-l (band V) and 1966 -1973 iire obtained from the fitting analyses independently of the lineshape model used.Only two or three bands are dominant in each spectrum. Compared to bands 1, I I and 111, the bands IV and V are assigned to correspond to a weaker polar contact bctween the CO ligand and a polar group in the heme pocket (probably Thr252) because of the opposite effcct of glycerol (osmotic pressure) and hydrostatic pressure on the inlensity 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 rolc for the substate equilibrium. This substate equilibrium freezes in at the glass transition temperature, Tg, of the proteinhlvent mixture. For the temperature region above T, the thermodynamic parameters and volumes for the substates have been delermined 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,,,,,.Keywords: cytochrome P-450 : infrared spectroscopy ; conformational substates.The CO stretch mode of the carbon monoxide complex of hemoproteins i s widely used as a sensitive spectroscopic probe to characterize the heme pockct. Electronic effects within the heme complex, proximal ligand charge, polar effects, and steric contraints as well as hydrogen bonding on the distal side are parameters that intluence the CO stretch frequency in hemoproteins (Ray et al., 1994: Springcr et al., 1994. CO stretch modes arc observed in most of the hemoproteins and are interpreted as indicating taxonomic conformational substales of thc hemoprotein (Frauenfelder et al., 1991: Jung and Marlow, 1987). Recently, wc have shown for camphor-bound cytochrome P-450,,,,,, ( P-4SOC,\,,,) that the CO stretch frequency is strongly intluenced by solvent parameters (pH, glycerol) and physical parameters (temperaturc and pressure) (Schulze et al., 1994). In addition, we have shown that small molecules, occupying the heme pocket, like substrates in cytochrome P-450 dramatically change the infrared spectrum (Jung et al., 1992a; Tsubaki et al., 1992 , 1995). A very complex behaviour of the infrared spectrum ic observed for the carbon monoxide complex of substrate-frcc cytochrome P-450,,,,, as will be demonstrated in this paper. MATERTALS AND METHODSProtein purification and sample preparation. Cytochrome P-450,,,,, from Pseudoinonas putirk exprcssed in Eschrrichia coli TB1 was isolated and purified according to Jung et al. (1992a) until an absorbance ratio 392 nmd28O nm of 1.3 was obtained.The procedurc for substrace removal followcd the or...

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The crystal structure of cytochrome P-450cam complexed with the enantiomer (lS)-camphor has been solved to 1.8 A resolution and compared with the structure of the (1R)-camphor P-450cam complex. The overall protein structure is the same for both enantiomer complexes. However, the orientation of the substrates in the heme pocket differs. In contrast to (1R)-camphor, the (lS)-enantiomer binds in at least two orientations. The major binding mode of (lS)-camphor resembles the one of the (lR)-enantiomer in that there is a hydrogen bond between Tyr-96 and the quinone group of camphor, and the 10-methyl group points towards the I-helix. The binding differs in that C-5 is not at a position suitable for hydroxylation. In the other orientation (lS)-camphor is not hydrogen bonded, but C-5 is located suitably for hydroxylation.

The effect of pH, monovalent cations, glycerol, temperature, and pressure on the carbonmonoxy (CO) stretching mode of camphor-bound cytochrome P-450,-(CYP 101) was studied. Two effects, band overlap and frequency shift, have been observed. The CO stretch infrared band located at about 1940 cm-I is asymmetric because of the overlap of three bands at about 1931 cm-I, 1939 cm-I, and 1942 cm-' with strongly different populations. Reducing the temperature or increasing the pressure leads to splitting the band or switching the asymmetry from the lower energy side to the higher energy side of the infrared band. The overlap of several CO stretch bands indicates conformational substates within the heme pocket. A frequency shift of the predominately populated band is observed by changing all the parameters mentioned. The pH-induced frequency shift follows an Sshape with the pK at 6.2, which matches the pK observed for the pH-induced high-spidow-spin transition. Conformational changes on the proximal heme side are suggested to be the origin. Monovalent cations at saturating concentration induce a small frequency shift depending on the ion radius. The potassium ion is the one that induces a CO stretch frequency with the highest wavenumber while sodium and lithium (smaller radii) and rubidium and caesium ion (larger radii) have diminished values, which is supporting evidence for the special function of the potassium ion within the structure. Glycerol and hydrostatic pressure induce a red shift of the CO stretching frequency. Forced contact of the polar hydroxyl group of Thr252 of the I helix induced by pressure and indirectly by glycerol is suggested to change the CO dipole moment, reflecting in the decreased CO stretching frequency.For many years, the CO stretching mode of the carbon monoxide complex of hemoproteins has been used as a sensitive spectroscopic probe for analysing the structure of the heme pocket. There are many papers which discuss the different structural parameters influencing the CO stretching frequency v(C0). Most of the important papers are covered in the recent article by Ray et al. (1994).Electronic effects within the heme complex, proximal ligand charge, polar effects, and steric constraints, as well as hydrogen bonding on the distal side, influence the CO stretching vibration frequency. Additionally, multiple CO infrared bands appear. It is very difficult to determine which of these parameters is most important in explaining the CO stretching mode in a particular hemoprotein. Care has to be taken in transferring conclusions drawn from one particular hemoprotein to another. For a detailed insight, the effect of solvent parameters and physical parameters has to be studied.The CO stretching mode as a spectroscopic probe is very helpful especially for cytochrome P-450 because it is sensitive to P-450-substrate interaction (Jung et al., 1992) and to Correspondence to C. Jung, Max Delbriick Centre for Molecular Medicine, Robert-Rossle-Str. 10, D-13122 Berlin, GermanyAbbreviations. P-450,,,, camphor-hydrox...

The CO stretch mode of various substrate complexes of cytochrome P-450cam-CO was measured using FT infrared spectroscopy. At room temperature most of the complexes show a single, but often asymmetric infrared band. The representative wavenumber of this band for the various complexes increases when the high-spin content, induced by the substrates in the oxidized protein, decreases. Additionally, the increase of the CO stretch wavenumber (1939 to 1956 cm-1) correlates with the decrease of the Soret band wavenumber (22440 to 22373 cm-1). It is suggested that the polarity of the heme pocket is modulated by the substrates due to changed accessibility of the heme environment for water molecules. The increased water content compensates positive electrostatic potentials near the CO ligand, which results in loosening the contact of CO to the I helix.