Substrate analog induced changes of the carbon-oxygen-stretching mode in the cytochrome P450cam-carbon monoxide complex

Abstract

The CO-stretching mode of the carbon monoxide ligand in reduced cytochrome P450cam, in the absence or presence of camphor and in the presence of nine different camphor analogues, was measured at room temperature using Fourier transform infrared spectroscopy. Substrate-free cytochrome P450cam--CO reveals a broad, slightly structured band resulting from an overlap of several stretching mode signals. The multitude of the signals indicates that cytochrome P450 exists in a dynamic equilibrium of several conformational substates. Binding of camphor or camphor analogues strongly influences this equilibrium. For substrate analogues which are not able to form a hydrogen bond to the hydroxyl group of tyrosine 96, the CO-stretching band is rather broad and asymmetric. In contrast, substrate analogues with one quinone group which form a hydrogen bond to the Tyr96 OH induce a shift and a sharpening of the CO-stretching mode band. For substrate analogues with two hetero groups, the infrared spectrum is slightly asymmetric or a minor band appears. Sterical hindrance, substrate mobility, and protein flexibility finally determine the position and width of the CO-stretching mode signals.

Compressibility of the Heme Pocket of Substrate Analogue Complexes of CytochromeP‐450cam‐CO

“…In addition, it was shown by Hui Bon Hoa and Marden (1982) that the spin transition of cytochrome P-450cam is extremely sensitive to physico-chemical conditions like pH, cations, temperature and pressure. A significant effect of substrate binding on the stretching mode of the CO ligand in cytochrome P-450cam-CO was recently shown in our laboratory (Jung et al, 1992a) and by Tsubaki and coworkers for cytochrome P-4SOscc (Tsubaki et al, 1992). Unno et al (1994) influence of various substrate analogues on the recombination kinetics of the photodissociated CO ligand at different pressures.…”

“…This assumption is supported by our results of infrared spectroscopic studies for the CO stretching mode showing a broad band (five overlapping bands) in the absence of substrate and a reduced number of bands or only one band in the presence of the different substrate analogues. This finding has been explained by different water contents (Jung et al, 1992a).…”

“…Cytochrome P -450cam from Pseudomona putidn (CYPI 01) was expressed in Escherichin coli strain TB 1, isolated and purified as described by Jung et al (1992a Fig. 1, 9) was depleted from the protein by dialysis of the concentrated cytochrome P-450 protein (approximately 1 mM) against 100 mM Tris/HCl pH 7, 20% glycerol, followed by a Sephadex (3-25 (medium) chromatography run in the same buffer and reconcentrated using the Amicon ultrafiltration cell to about 0.535 mM (protein stock solution).…”

Tyrosine Radical Formation in the Reaction of Wild Type and Mutant Cytochrome P450cam with Peroxy Acids

“…Cells were grown in standard LB medium in a 22l fermentor as described previously (18) or in flasks.…”

“…Protein Purification-The purification of the expressed wild type and mutant proteins followed the same protocol (18). The final purification step of the proteins was performed with fast protein liquid chromatography-anion exchange chromatography to get an absorbance ratio Q ϭ 392 nm/280 nm of Ͼ1.4 for 100% high spin state content.…”