The Carbon Monoxide Stretching Modes in Camphor‐Bound Cytochrome P‐450_cam

Abstract

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...

Substrate binding to cytochromes P450

“…For these studies, the Soret band [151, 152] and the stretching mode vibration of CO bound to the reduced enzyme (monitored by IR spectroscopy) are utilized as spectral probes [153]. Also, flash photolysis [154, 155] and stopped-flow [150, 156] techniques have been employed under high pressure in order to investigate the steps involved in binding of substrates.…”

Substrates modulate the rate‐determining step for CO binding in cytochrome P450cam (CYP101)

“…An indirect method is a water replacement technique using a probe molecule. In a large number of studies [4–9] using different approaches we found that the CO iron ligand is a good probe for the polarity and therefore for the presence of water molecules in the heme environment of cytochrome P450cam.…”

Time‐resolved Fourier‐transform infrared studies of the cytochrome P‐450cam carbonmonoxide complex bound with (1R)‐camphor and (1S)‐camphor substrate

“…The spectra obtained in this way correspond to an absorbance difference spectrum between the absolute spectrum of the CO bound form in the dark (so-called dark-spectrum) and the absolute spectrum of the photolyzed sample (so-called light-spectrum). For the darkspectrum of (1R)-camphor-bound P-450 we have shown elsewhere [12] that the infrared band is asymmetric which results from an overlap of the subconformer bands. Similar behavior is observed also for (1S)-camphor-bound P-450¢~m CO, however, with different shape (Schulze, H., private communication).…”