Rates of heme oxidation and reduction in Ru(His33)cytochrome c at very high driving forces

The rates of Ru(His33)cytochrome c electron-transfer (ET) reactions have been measured over a driving-force range of 0.59 to 1.89 eV. The driving-force dependence of Fe²⁺ → Ru³⁺ ET in RuL₂(im)(His33)cyt c [L = 2,2'-bipyridine (bpy), 4,4',5,5'-tetramethyl-2,2'-bipyridine (4,4',5,5'-(CH₃)₄-bpy), 4,4'-dimethyl-2,2'-bipyridine (4,4'-(CH₃)₂-bpy), 4,4'-bis(N-ethylcarbamoyl)-2,2'-bipyridine (4,4'-(CONH(C₂H₅))₂-bpy), 1,10-phenanthroline (phen); im = imidazole] is well described by semiclassical ET theory with k(max) = 2.7 x 10⁶ s^-1 (H(AB) = 0.095 cm^-1) and λ = 0.74 eV. As predicted by theory, the rate of an exergonic (-ΔG° = 1.3 eV) heme reduction reaction, *Ru²⁺(bpy)₂(im)(His) → Fe³⁺, falls in the inverted region (k = 2.0 x 10⁵ s^-1). In contrast, the rates of three highly exergonic heme reductions, *Ru²⁺(phen)₂(CN)(His) → Fe³⁺ (2.0 x 10⁵ s^-1; 1.40 eV), Ru⁺(4,4'-(CONH(C₂H₅))₂-bpy)₂(im)(His) → Fe³⁺ (2.3 x 10⁵ s^-1; 1.44 eV), and Ru⁺(phen)₂(CN)(His)→ Fe³⁺ (4.5 x 10⁵ s^-1; 1.89 eV), are much higher than expected for reactions directly to ground-state products. Agreement with theory is greatly improved by assuming that an electronically excited ferroheme (Fe²⁺ → *Fe²⁺; ~1.05 eV) is the initial product in each of these reactions.