26] Detection and quantitation of free cytochrome P-450 and cytochrome P-450 complexes by EPR spectroscopy

Abstract

Publisher Summary This chapter describes the detection and quantitation of free cytochrome P-450 and cytochrome P-450 complexes by electron paramagnetic resonance (EPR) spectroscopy. Cytochromes P-450 in the oxidized state exhibit EPR spectra at low temperature, both in the free form and when bound to substrates, intermediates and products, or certain inhibitors. The EPR spectra fall into two categories, those arising from a high-spin heme species with absorption maxima at approximately g = 8, 4, and 1.8 and those arising from a low-spin heme species (S = 1/2) with absorption maxima at approximately g = 2.4, 2.25, and 1.9; which form is observed depends on the state of ligation of the cytochrome. EPR spectra of high-spin P-450 are not generally observed above 20°K. This species is not easily saturable as the low-spin form, and powers in the mW region may often be used. There is an additional problem related to the zerofield splitting in the case of high-spin heme as opposed to low-spin heme.