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

The kinetics of oxyferryl (Fe IV O) heme reduction in horse heart myoglobin (Mb) by a 4 LRu II (a = NH 3; L = NH 3, pyridine, isonicotinamide) bound at the surface His48 were investigated with pulse radiolysis. The observed first-order rate constants ( k obs1 ) decreased with increasing pH and reduction potential for the a 4 LRu centers ( E °/Ru III/II = 77, 330, and 400 mV for L = NH 3, Pyr, and Isn). Rate−pD data obtained in D 2 O for the a 5 Ru derivative revealed the presence of an equilibrium isotope effect, and a p K a of 5.7 (6.2 in D 2 O) 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 a 4 LRu derivatives. Marcus theory analysis of the k ET (0.74, 1.8, and 3.6 s - 1 for L = Isn, Pyr, and NH 3 ) extracted from the k obs1 values yielded a reorganization energy (λ) of 1.8 for Ru II → Fe IV O ET in the a 4 LRu derivatives but a λ of 2.1 eV for the a 5 Ru derivative. From the latter, it is concluded that ET is strongly gated in the a 5 Ru derivative, and this is assumed to be the major reason for the low reactivity of Fe IV O in Mb at high −Δ G °.