PH and Driving Force Dependence of Intramolecular Oxyferryl Heme Reduction in Myoglobin

Abstract

The kinetics of oxyferryl (FeIVO) heme reduction in horse heart myoglobin (Mb) by a4LRuII (a = NH3; L = NH3, pyridine, isonicotinamide) bound at the surface His48 were investigated with pulse radiolysis. The observed first-order rate constants (kobs1) decreased with increasing pH and reduction potential for the a4LRu centers (E°/RuIII/II = 77, 330, and 400 mV for L = NH3, Pyr, and Isn). Rate−pD data obtained in D2O for the a5Ru derivative revealed the presence of an equilibrium isotope effect, and a pKa of 5.7 (6.2 in D2O) was obtained for the acid−base group, which is assigned to the distal His64. A mechanism where protonation precedes ET provides a good fit of the kinetic data for the three a4LRu derivatives. Marcus theory analysis of the kET (0.74, 1.8, and 3.6 s-1 for L = Isn, Pyr, and NH3) extracted from the kobs1 values yielded a reorganization energy (λ) of 1.8 for RuII → FeIVO ET in the a4LRu derivatives but a λ of 2.1 eV for the a5Ru derivative. From the latter, it is concluded that ET is strongly gated in the a5Ru derivative, and this is assumed to be the major reason for the low reactivity of FeIVO in Mb at high −ΔG°.