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

Heme oxygenase (HO) catalyzes the O₂- 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 α-meso-hydroxyheme. We employed EPR (using both ¹⁶O₂ and ¹⁷O₂) and ¹H, ¹⁴N ENDOR spectroscopies to characterize the intermediates generated by 77 K radiolytic cryoreduction and subsequent annealing of wild-type oxy-HO and D140A, F mutants. One-electron cryoreduction of oxy-HO yields a hydroperoxoferri-HO with g-tensor, g = [237, 2,187, 1.924]. Annealing of this species to 200 K is accompanied by spectroscopic changes that include the appearance of a new ¹H ENDOR signal, reflecting rearrangements in the active site. Kinetic measurements at 214 K reveal that the annealed hydroperoxoferri-HO species, denoted R, generates the ferri-α-meso-hydroxyheme product in a first-order reaction. Disruption of the H-bonding network within the distal pocket of HO by the alanine and phenylalanine mutations of residue D140 prevents product formation. The hydroperoxoferri-HO (D140A) instead undergoes heterolytic cleavage of the O-O bond, ultimately yielding an EPR-silent compound II-like species that does not form product. These results, which agree with earlier suggestions, establish that hydroperoxoferri-HO is indeed the reactive species, directly forming the α-meso-hydroxyheme product by attack of the distal OH of the hydroperoxo moiety at the heme α-carbon.