Linear Electric Field-Induced Shifts in Electron Paramagnetic Resonance: A New Method for Study of the Ligands of Cytochrome P-450

Abstract

The linear electric field shift in paramagnetic resonance has, for the first time, been observed in frozen solutions. The magnitude of the shift parameters, measured at g.. of the electron paramagnetic resonance absorption envelope,_ has been used to characterize lowspin ferric heme mercaptide complexes where the nonmercaptide liganid was varied. The magnitude of the shift provides a measure of the difference between the crystal field contribution of the mercaptide and nonmercaptide ligands. In low-spin ferric cytochrome P-450 of rat liver, the presence of an electric field shift indicates a deviation from inversion symmetry and proves that the axial ligands to the heme are not the same. From the magnitude of the shift and its dependence upon the angle between applied electric and magnetic fields, it is suggested that the nonaxial ligand to the heme has a crystal field contribution greater than imidazole and smaller than guanidine.Cytochrome P450 represents a class of heme proteins characterized by an unusual optical absorption when reduced and reacted with CO (1, 2). Various members of this class have been isolated from bacteria (3, 4) and mammals (5, 6), but cytochrome P450 found in the endoplasmic reticulum of mammalian liver [and which is the terminal oxidase of the xenobiotic hydroxylating systems (7) ] has drawn the greatest attention.In liver slices and liver microsomes, cytochrome P450 is found largely in the low-spin ferric state and can therefore be studied by electron paramagnetic resonance (EPR) spectroscopy (7,8). On the basis of magnetic resonance comparison with model compounds, it has been suggested that a necessary z ligand to heme of all cytochromes P450, irrespective of their sources, is a mercaptide sulfur, endogenous to the protein structure and most likely from a cysteinyl residue (9, 10). The nature of the nonmercaptide z ligand in the various cytochromes P450 remains unknown, although EPR examination suggests a heterogeneity of structures, at least in soluble preparations from liver microsomes (11, 12).It is, however, the presence of the mercaptide ligand in cytochrome P450 that essentially determines the EPR behavior. It is difficult to make structural assignments on the basis of EPR, as was done for various low-spin heme proteins (9, 13), since the second z ligand has only a minor effect on the g tensor.In this preliminary report we describe a new technique that makes it possible to investigate cases of this kind. Specifically, we have observed for the first time a linear electric fieldinduced shift in the EPR frequency of low-spin ferric cytochrome P-450 from rat-liver microsomes of a phenobarbitaltreated animal and also of several heme mercaptide model compounds in frozen solution. In this way we were able to characterize the deviations from inversion symmetry of the center composed of the heme group and the ligands in these materials and to show that there is a marked difference in the bonding strengths of the two z ligands in the heme protein. In consideration of the magnitu...

Pulsed electron paramagnetic resonance studies of the copper(II) site in galactose oxidase

“…Measurements of the electron spin-echo envelope were made as described by Mims & Peisach (1976a), and the LEFE measurements were made as described by Peisach & Mims (1973) and Mims (1974). The echo envelopes obtained in three-pulse experiments were Fourier transformed as described previously (Shimizu et al, 1979;.…”

Pulsed electron paramagnetic resonance studies of types I and II copper of Rhus vernicifera laccase and porcine ceruloplasmin

“…Recordings of the echo amplitude function were obtained as described in a previous paper (Mims and Peisach, 1976a). For this study, microwave pulses were about 20 ns long, microwave power levels were about 200 W, the integration time constant of the boxcar circuitry was 0.5 ms, and the time required to trace the echo envelope was 400 s. The LEFE was studied using the signal half-fall procedure (Peisach and Mims, 1973). Concentration of proteins used varied from 3 to 5 mM as judged from published extinction coefficients near 600 nm (Nakamura and Ogura, 1966;Blumberg et al, 1963).…”

“…Nuclear modulation patterns show that imidazole is a ligand for all of them. The linear electric field effect (LEFE) in EPR was It has recently been shown that pulsed EPR1 techniques provide a useful means for assigning ligands (Peisach and Mims. 1973;Mims and Peisach, 1976a) and for investigating f From the Institute of Applied Biochemistry, University of Rome and CNR Center for Molecular Biology, Rome 00185, Italy (B.M.…”

“…This effect is termed the "nuclear modulation effect". A second type of experiment, also involving the generation of electron spin-echoes, is concerned with the measurement of the g shifts induced by the application of an external electric field (Peisach and Mims, 1973;Peisach, 1974, 1976b). These "linear electric field effect" experiments yield information about the odd symmetry part of the ligand field 1 Abbreviations used are: EPR, electron paramagnetic resonance; LEFE, linear electric field effect.…”

Electrically Induced Crystal Field Distortion in a Ferroelectric Perovskite Revealed by Electron Paramagnetic Resonance

“…In the early years, the investigations of the E-field effect of spins by techniques including electron paramagnetic resonance (EPR) mainly focus on determining the symmetry of transition metal centers’ coordination environment. , More recently, this related technology is employed to study the SEC of electron spins and to prove the possibility of manipulating electron spins in quantum information processing. ,,− , In a pulsed EPR experiment, the quantum phase of the superposition state of an electron spin will evolve under an E-field pulse, the rate of which is an indicator for the strength of the SEC. While in continuous wave (CW)-EPR experiments, the SEC effect can be detected with E-field modulation (EFM) instead of the magnetic field modulation (MFM) in the standard CW-EPR setup. , Compared to pulsed experiments, EFM-EPR has the advantages of easy operation and not being limited by the phase memory time, providing us a powerful method of SEC measurement.…”