Excitation wavelength dependence of photoreduction of cytochrome c and its mechanism monitored by resonance Raman spectroscopy

Purified monomeric cytochrome c (Cyt-c) at neutral pH exhibited extensive photoreduction under anaerobic conditions (a fore pressure of 10(-5) Torr) upon laser irradiation in the 400-450 nm region and the resonance Raman (RR) spectrum of the photoreduced species was identical with that of the chemically reduced species. However, only partial photoreduction occurred at pH 10.1 where the axial methionine ligand is replaced probably by lysine, and no photoreduction took place after the transitions with pK(a) = 12.76 and 2.5 where the axial histidine ligand is replaced by other residue. The photoreduction was completely inhibited in the presence of oxygen suggesting active participation of a triplet state in this process. The relative quantum yield for photoreduction of Cyt-c at neutral pH as a function of excitation wavelengths shows a weak maximum centered around 410 nm while it increases almost exponentially when excitation wavelength is moved from 380 to 330 nm and then tends to saturate from 330 to 260 nm. Immediate electron donors to the heme and plausible mechanisms of the photoreduction are discussed. We have also investigated the effects laser power, salt and buffer concentration on the yield of photoreduction.