Conformational substates of the oxyheme centers in alpha and beta subunits of hemoglobin as disclosed by EPR and ENDOR studies of cryoreduced protein.

Exposure of frozen solutions of oxyhemoglobin to gamma-irradiation at 77 K yields EPR- and ENDOR-active, one-electron-reduced oxyheme centers which retain the conformation of the diamagnetic precursor. EPR spectra have been collected for the centers produced in human HbO(2) and isolated alphaO(2) and betaO(2) chains, as well as alphaO(2)beta(Zn), alpha(Zn)betaO(2), and alphaO(2)beta(Fe(3+)) hybrids, each in frozen buffer and in frozen glasses that form in the presence of glycols and sugars and also in the presence of IHP. These reveal two spectroscopically distinct classes of such ferriheme centers (g(1) View Article and Find Full Text PDF

Catalytic mechanism of heme oxygenase through EPR and ENDOR of cryoreduced oxy-heme oxygenase and its Asp 140 mutants.

Heme oxygenase (HO) catalyzes the O(2)- and NADPH-cytochrome P450 reductase-dependent conversion of heme to biliverdin, Fe, and CO through a process in which the heme participates both as a prosthetic group and as a substrate. In the present study, we have generated a detailed reaction cycle for the first monooxygenation step of HO catalysis, conversion of the heme to alpha-meso-hydroxyheme. We employed EPR (using both (16)O(2) and (17)O(2)) and (1)H, (14)N ENDOR spectroscopies to characterize the intermediates generated by 77 K radiolytic cryoreduction and subsequent annealing of wild-type oxy-HO and D140A, F mutants.