Flavins of NADPH-cytochrome P-450 reductase: evidence for structural alteration of flavins in their one-electron-reduced semiquinone states from resonance Raman spectroscopy.

Abstract

The mechanism of electron transfer from NADPH to cytochrome P-450 through FAD and FMN of the reductase is largely unknown. In this paper, we report the resonance Raman spectral properties of the oxidized and the semiquinonoid states of the flavins in the holoenzyme and the FMN-depleted forms, respectively, of detergent-solubilized rabbit liver microsomal NADPH-cytochrome P-450 reductase. The resonance Raman spectra of the oxidized forms [FAD; FMN] and [FAD;-] were essentially identical, indicating similar binding interactions of these flavins with the protein. To the contrary, the spectra of the semiquinonoid FADH. and FMNH. forms revealed significant spectral differences. Both O2-unstable species, characterized as [FADH.; FMNH2] and [FADH.;-] excited at 568.2 nm, have dominant spectral peaks at approximately 1611, 1539-1543, 1377, 1305, 1263, and 1226 cm-1. However, in the O2-stable [FAD; FMNH.] species, resonance Raman bands were located at 1611, 1532, 1388, 1304, 1268, and 1227 cm-1 when excited at the same wavelength. The approximately 10-cm-1 shifts of the 1532- and 1388-cm-1 bands suggest that the environments surrounding rings II and III of the isoalloxazines change upon reduction to semiquinonoid forms. It is proposed that N1 of FADH. (as a hydrogen-bond acceptor) and N5 of FMNH. (as donor) provide the distinguishing flavin-protein interactions in the semiquinonoid states. Furthermore, the resonance Raman spectra of the semiquinonoid species appear to be missing a number of bands assigned to ring I vibrations in the spectra of the oxidized flavins.(ABSTRACT TRUNCATED AT 250 WORDS)