Transient and Time-Resolved Resonance Raman Investigation of Photoinitiated Electron Transfer in Ruthenated Cytochromes c

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Abstract
Ruthenation of exterior amino acid residues of heme proteins provides an effective means by which biological ET reactions can be studied within the context of highly complex protein environments. Resonance Raman spectroscopy can probe both ET kinetics and structural dynamics at the molecular level. Here we present the first comprehensive use of time-resolved and transient resonance Raman spectroscopies to examine photoinduced ET in cytochromes. Two ruthenated cytochromes c, Ru(lys72)cyt.c and Ru(cyt102)cyt.c, were studied with TRRS using 10 ns laser pulses and with TRRRS on a 10 ns to 10 ms time scale. It was found that resonance Raman protocols can effectively trace ET kinetics and associated heme−protein structural dynamics. Care must be exercised, however, when drawing comparisons to measurements made by other methods (i.e., transient absorbance). The TRRS studies directly probe the heme and its local environment and reveal that the heme dynamics accompanying ET are very rapid relative to phenomenological ET kinetics. The heme and its local environment evolve to their equilibrium (ferrous) structure in less than 10 ns subsequent to ET, with no evidence for the existence of metastable heme pocket geometries analogous to those observed in the dynamic response of hemoglobins and oxidases. Finally, species-specific differences are observed in the photoinduced ET kinetics and heme structural dynamics. However, these differences are confined to nanosecond or faster time scales.