Jörg Contzen

Spectroscopic characterization of the iron-oxo intermediate in cytochrome P450

Abstract From analogy to chloroperoxidase from Caldariomyces fumago, it is believed that the electronic structure of the intermediate iron-oxo species in the catalytic cycle of cytochrome P450 corresponds to an iron(IV) porphyrin-π-cation radical (compound I). However, our recent studies on P450cam revealed that after 8 ms a tyrosine radical and iron(IV) were formed in the reaction of ferric P450 with external oxidants in the shunt pathway. The present study on the heme domain of P450BM3 (P450BMP) shows a similar result. In addition to a tyrosine radical, a contribution from a tryptophan radical was found in the electron paramagnetic resonance (EPR) spectra of P450BMP. Here we present comparative multi-frequency EPR (9.6, 94 and 285 GHz) and Mössbauer spectroscopic studies on freeze-quenched intermediates produced using peroxy acetic acid as oxidant for both P450 cytochromes. After 8 ms in both systems, amino acid radicals occurred instead of the proposed iron(IV) porphyrin-π-cation radical, which may be transiently formed on a much faster time scale. These findings are discussed with respect to other heme thiolate proteins. Our studies demonstrate that intramolecular electron transfer from aromatic amino acids is a common feature in these enzymes. The electron transfer quenches the presumably transiently formed porphyrin-π-cation radical, which makes it extremely difficult to trap compound I.

TIME-RESOLVED FOURIER-TRANSFORM INFRARED STUDIES OF THE CYTOCHROME P-450CAM CARBONMONOXIDE COMPLEX BOUND WITH (1R)-CAMPHOR AND (1S)-CAMPHOR SUBSTRATE

The CO‐binding reaction of cytochrome P‐450cam bound with (1R)‐camphor and (1S)‐camphor are compared in the temperature region of 210–260 K using time‐resolved Fourier‐transform infrared spectroscopy with the CO stretch vibration as spectroscopic probe. For (1S)‐camphor as substrate the association of CO is slowed down by a factor of 2, while the dissociation is accelerated by a factor of 3. The CO complex for the (1S)‐camphor‐bound P‐450 is less stabilized (ΔG=−22 kJ/mol) compared to the natural substrate (1R)‐camphor (ΔG=−30 kJ/mol). The data are interpreted by a smaller change of the mobility of the (1S)‐camphor due to CO binding as compared to (1R)‐camphor, which would indicate a higher mobility of (1S)‐camphor already in the CO free reduced form of P‐450cam. The higher mobility of (1S)‐camphor in the heme pocket might explain the increased uncoupling rate (hydrogen peroxide formation) of 11% [Maryniak et al. (1993) Tetrahedron 49, 9373–9384] during the P‐450cam catalyzed hydroxylation compared to 3% for the conversion of (1R)‐camphor.

Intermolecular electron transfer in cytochrome P450cam covalently bound with Tris(2,2′-bipyridyl)ruthenium(II): structural changes detected by FTIR spectroscopy

Using Fourier transform infrared spectroscopy (FTIR) we have monitored the changes in the protein structure following photoinduced electron transfer from Ru(bpy)(3)(2+) covalently attached to cysteine 334 on the surface of cytochrome P450cam (CYP101). The FTIR difference spectra between the oxidized and reduced form indicate changes in a salt link and the secondary structure (alpha-helix and turn regions). Photoreduction was carried out in the presence of carbon monoxide in order to prove the reduction of the heme iron by means of the appearance of the characteristic CO stretch vibration infrared band at 1940 cm(-1) for the camphor-bound protein. This infrared band has also been used to estimate electron transfer rates. The observed rates depend on the protein concentration, indicating that intermolecular electron transfer occurs between the labeled molecules.