Transient and Stationary Spectroscopy of Cytochrome c: Ultrafast Internal Conversion Controls Photoreduction


* (Karl Kleinermanns); (Sergey Kovalenko)


Photoreduction of cytochrome c (Cyt c) has been reinvestigated using femtosecond-to-nanosecond transient absorption and stationary spectroscopy. Femtosecond spectra of oxidized Cyt c, recorded in the probe range 270–1000 nm, demonstrate similar evolution upon 266 or 403 nm excitation: an ultrafast 0.3 ps internal conversion followed by a 4 ps vibrational cooling. Late transient spectra after 20 ps, from the cold ground-state chromophore, reveal a small but measurable signal from reduced Cyt c. The yield φ for Fe3+→Fe2+ photoreduction is measured to be φ403 = 0.016 and φ266 = 0.08 for 403 and 266 nm excitation. These yields lead to a guess of the barrier EfA = 55 kJ mol−1 for thermal ground-state electron transfer (ET). Nanosecond spectra initially show the typical absorption from reduced Cyt c and then exhibit temperature-dependent sub-microsecond decays (0.5 μs at 297 K), corresponding to a barrier EAb = 33 kJ mol−1 for the back ET reaction and a reaction energy ΔE = 22 kJ mol−1. The nanosecond transients do not decay to zero on a second time scale, demonstrating the stability of some of the reduced Cyt c. The yields calculated from this stable reduced form agree with quasistationary reduction yields. Modest heating of Cyt c leads to its efficient thermal reduction as demonstrated by differential stationary absorption spectroscopy. In summary, our results point to ultrafast internal conversion of oxidized Cyt c upon UV or visible excitation, followed by Fe-porphyrin reduction due to thermal ground-state ET as the prevailing mechanism.