Catalytic Mechanism of Heme Oxygenase through EPR and ENDOR of Cryoreduced Oxy-Heme Oxygenase and Its Asp 140 Mutants

Abstract

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. One-electron cryoreduction of oxy-HO yields a hydroperoxoferri-HO with g-tensor, g = [2.37, 2.187, 1.924]. Annealing of this species to 200 K is accompanied by spectroscopic changes that include the appearance of a new (1)H 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-alpha-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 alpha-meso-hydroxyheme product by attack of the distal OH of the hydroperoxo moiety at the heme alpha-carbon.

Crystal Structure of the Dioxygen-bound Heme Oxygenase from Corynebacterium diphtheriae

“…DISCUSSION HmuO, like HO-1, yields only the ␣-isomer of biliverdin (12). Regioselectivity of HO catalysis is defined at the first oxygenation step (50), where the remote hydroperoxo oxygen attacks the porphyrin ␣-meso-carbon to form ␣-meso-hydroxyheme (6,25). Previous studies have suggested that regiospecific degradation of heme by HO activity is primarily under the steric control.…”

“…3). Asp-136, which is equivalent to the catalytically critical Asp-140 of HO-1 (25,48,49), stabilizes Wat1 and Wat2. Wat2 is connected by Wat3 and Wat4 to the side chain of Gln-46, which, in turn, is connected by another water molecule to the surface amino acid, Asp-86.…”

“…This internal water molecule is part of a hydrogen-bonding network that extends from the Fe-O 2 unit to solvent water. This network provides a channel for efficient proton transfer to the active site, which is required for formation of the ferric hydroperoxo species after a one-electron reduction of the oxy form, activation of the O-O bond for cleavage, and hydroxylation of the ␣-meso-carbon (25,48,49). The second water molecule (Wat2) of the network is hydrogen bonded to Asp-136.…”

“…These results, which indicated both steric restriction and hydrogen bonding, led to the proposal that the terminal oxygen is "held" in a position adjacent to the ␣-mesocarbon of the porphyrin ring, facilitating a highly regiospecific oxygenation (14,20,24); but, again, this orientation has not been confirmed directly by x-ray crystallography. A hydrogen bond with an adjacent exchangeable proton from a distal pocket water molecule has also been proposed (13,22), and this proton is thought to become the hydroperoxo proton after a one-electron reduction of the oxy form (17,25). This hydrogen bonding proposal was based on electron paramagnetic resonance studies with HmuO and HO containing oxy cobalt porphyrin and also requires direct structural confirmation.…”

Reaction mechanisms of 15-hydroperoxyeicosatetraenoic acid catalyzed by human prostacyclin and thromboxane synthases

“…Cpd I has an EPR signal at g= 2, whereas Cpd II is EPR silent [28,29]. For PGIS, approximate 16% of the low-spin heme signals were retained upon reaction with 15-HPETE (solid line vs. dotted line in Fig.…”