Photoionization of ferrocytochrome c by 248 nm laser light and the observation of the early stages of ferricytochrome c unfolding in the nanosecond-to-millisecond timescale

Photolysis of ferrocytochrome c by 248 nn laser light in aqueous solution a t pH 7 generates hydrated electrons (e(aq)(-)) by a monophotonic process wi th quantum yield phi = 0.034 Approximately three-quarters of the e(aq)(-) o riginate from the heme, which is converted from the ferrous to the ferric s tate in <100 ns, The conformational changes associated with the change in t he redox state of cytochrome c are either not detectable spectrophotometric ally or complete in <100 ns, Also, under conditions where ferrocytochrome c is stable but ferricytochrome c is unfolded (3 M guanidine, pH 7, 40 degre es C), photoionization of ferrocytochrome c generated ferricytochrome c wit h similar quantum yield. Under these conditions, the lifetime of native fer ricytochrome c is 67 mu s; it decays via two intermediates with lambda(max) > 410 nm, neither of which is the thermodynamically favored, unfolded form . These species are putatively identified as unfolding intermediates with n onnative iron ligands, similar to those found during folding of ferrocytoch rome c, The results suggest that unfolding, like folding, proceeds by intra chain diffusion and ligand exchange.