Time-resolved resonance Raman spectroscopy of cytochrome c reduced by pulse radiolysis.

The first investigation of the dynamics of a redox transition of an electron-transfer enzyme by time-resolved resonance Raman spectroscopy in combination with pulse-radiolytic reduction is described by an application to cytochrome c. A long-lived transient state is observed upon reduction of the alkaline form of cytochrome c as a distinct frequency shift of one resonance Raman band. From the frequency in the stable oxidized state, 1567 cm(-1), this particular resonance Raman band shifts within less than 1 microsecond to 1533 cm(-1) in the transient reduced state, which has a lifetime longer than 20 ms but shorter than a few seconds. Finally, in the stable reduced state, this band is located at 1547 cm(-1). According to a previous normal coordinate analysis, this resonance Raman band can be assigned predominantly to a stretching mode of the outermost C-C bonds in the four pyrrole rings of porphyrin. This vibrational mode is influenced by the protein most directly through the covalent thioether linkages of two cysteines to porphyrin. We interpret the long lifetime of the transient state as due to the slow return of Met-80 as sixth ligand to the heme iron upon reduction of the alkaline form of cytochrome c.