Redox Reactivity of the Heme Fe3+/Fe2+ Couple in Native Myoglobins and Mutants with Peroxidase-like Activity

The reaction enthalpy and entropy for the oneelectron
reduction of the ferric heme in horse heart and
sperm whale aquometmyoglobins (Mb) have been determined
exploiting a spectroelectrochemical approach. Also
investigated were the T67R, T67K, T67R/S92D and T67R/
S92D Mb-H variants (the latter containing a protoheme-Lhistidine
methyl ester) of sperm whale Mb, which feature
peroxidase-like activity. The reduction potential (E¢) in all
species consists of an enthalpic term which disfavors Fe3+
reduction and a larger entropic contribution which instead
selectively stabilizes the reduced form. This behavior differs
from that of the heme redox enzymes and electron
transport proteins investigated so far. The reduction thermodynamics
in the series of sperm whale Mb variants
show an almost perfect enthalpy–entropy compensation,
indicating that the mutation-induced changes in
DH0
rc and DS0
rc are dominated by reduction-induced solvent
reorganization effects. The modest changes in E¢
originate from the enthalpic effects of the electrostatic
interactions of the heme with the engineered charged
residues. The small influence that the mutations exert on
the reduction potential of myoglobin suggests that the increased
peroxidase activity of the variants is not related to
changes in the redox reactivity of the heme iron, but are
likely related to a more favored substrate orientation within
the distal heme cavity.