Electron Paramagnetic Resonance and Electron-NuclearDouble Resonance Studies of the Reactions of Cryogenerated Hydroperoxoferric–HemoproteinIntermediates

摘要

The fleeting ferric peroxo and hydroperoxo intermediates of dioxygen activation by hemoproteins can be readily trapped and characterized during cryoradiolytic reduction of ferrous hemoprotein–O2 complexes at 77 K. Previous cryoannealing studies suggested that the relaxation of cryogenerated hydroperoxoferric intermediates of myoglobin (Mb), hemoglobin, and horseradish peroxidase (HRP), either trapped directly at 77 K or generated by cryoannealing of a trapped peroxo-ferric state, proceeds through dissociation of bound H2O2 and formation of the ferric heme without formation of the ferryl porphyrin π-cation radical intermediate, compound I (Cpd I). Herein we have reinvestigated the mechanism of decays of the cryogenerated hydroperoxyferric intermediates of α- and β-chains of human hemoglobin, HRP, and chloroperoxidase (CPO). The latter two proteins are well-known to form spectroscopically detectable quasistable Cpds I. Peroxoferric intermediates are trapped during 77 K cryoreduction of oxy Mb, α-chains, and β-chains of human hemoglobin and CPO. They convert into hydroperoxoferric intermediates during annealing at temperatures above 160 K. The hydroperoxoferricintermediate of HRP is trapped directly at 77 K. All studied hydroperoxoferricintermediates decay with measurable rates at temperatures above 170K with appreciable solvent kinetic isotope effects. The hydroperoxoferricintermediate of β-chains converts to the S =3/2 Cpd I, which in turn decays to an electron paramagnetic resonance(EPR)-silent product at temperature above 220 K. For all the otherhemoproteins studied, cryoannealing of the hydroperoxo intermediatedirectly yields an EPR-silent majority product. In each case, a secondfollow-up 77 K γ-irradiation of the annealed samples yieldslow-spin EPR signals characteristic of cryoreduced ferrylheme (compoundII, Cpd II). This indicates that in general the hydroperoxoferricintermediates relax to Cpd I during cryoanealing at low temperatures,but when this state is not captured by reaction with a bound substrate,it is reduced to Cpd II by redox-active products of radiolysis.