Contributions to cytochrome c inner- and outer-sphere reorganization energy

Cytochromes c are small water-soluble proteins that catalyze electron transfer in metabolism and energy conversion processes. Hydrogenobacter thermophilus cytochrome c₅₅₂ presents a curious case in displaying fluxionality of its heme axial methionine ligand; this behavior is altered by single point mutation of the Q64 residue to N64 or V64, which fixes the ligand in a single configuration. The reorganization energy (λ) of these cytochrome c₅₅₂ variants is experimentally determined using a combination of rotating disc electrochemistry, chronoamperometry and cyclic voltammetry. The differences between the λ determined from these complementary techniques helps to deconvolute the contribution of the active site and its immediate environment to the overall λ (λ_Total). The experimentally determined λ values in conjunction with DFT calculations indicate that the differences in λ among the protein variants are mainly due to the differences in contributions from the protein environment and not just inner-sphere λ. DFT calculations indicate that the position of residue 64, responsible for the orientation of the axial methionine, determines the geometric relaxation of the redox active molecular orbital (RAMO). The orientation of the RAMO with respect to the heme is key to determining electron transfer coupling (H_AB) which results in higher ET rates in the wild-type protein relative to the Q64V mutant despite a 150 mV higher λ_Total in the former.

Efficient delocalization of the redox-active molecular orbital (RAMO) in HtWT results in an increase in H_AB value which in turn accelerates the electron transfer (ET) rate in spite of the higher reorganization energy (λ) than the HtQ64V mutant.