MAGNETIC CIRCULAR-DICHROISM OF PURIFIED FORMS OF RABBIT LIVER CYTOCHROMES P-450 AND P-420

Magnetic circular dichroism (MCD) studies of three different forms of rabbit liver cytochrome P-450 are described. The cytochromes were isolated and purified to apparent homogeneity using procedures developed by D. A. Haugen and M. J. Coon [(1976), J. Biol. Chem. 251, 7929], The form of P-450 studied most extensively, PB-P-450-LM2, was shown to be enzymatically active. The major findings are: (1) The three forms of oxidized P-450 (PB-P-450-LM2, PB-P-450-LM3:4, and BNF-P-450-LM4) as isolated show substantially different MCD spectra. These differences are interpreted as being due to changes in the spin states of the heme iron. (2) The MCD spectra of the three forms in the reduced state as well as their carbonmonoxy derivatives are on the whole similar to each other. (3) Conversion to P-420 leads to MCD spectra which are substantially different from the corresponding P-450 spectra. The spectra of the reduced and reduced + CO forms of P-420 are similar to those of deoxy-and carbonmonoxymyoglobin, respectively. (4) Addition of an excess of the type I substrate d-benzphetamine to the predominantly low-spin oxidized form of P-450 causes conversion to an approximately equal mixture of high- and low-spin forms. (5) Computer fitting of the MCD spectra of mixed spin-state samples of oxidized P-450 has been performed using library spectra of P-450 in both pure spin states, thereby permitting accurate determinations of the spin-state composition of samples at ambient temperature. © 1978, American Chemical Society. All rights reserved.