| One of the unique features of enzymatic heme degradation is its regioselective hydroxylation that converts the ferric hydroperoxide intermediate to the alpha-meso-hydroxy-heme. In this research, we have studied the effect of some conserved amino acid residues in heme oxygenase (HO) on dictating the regioselectivity of bacterial heme oxygenation using recombinant DNA technique and spectroscopic methods. The results show that the regioselectivity is governed by in-plan heme orientation, which in turn is controlled by the interactions between the amino acid side chains and heme propionates. When the interactions are disrupted by mutating these conserved amino acid residues, including Arg177 in cd-HO, Lys16 and Tyr112 in nm-HO, Lys34 and Lys132 in pa-HO, heme in-plan rotation is observed. Consequently, a different meso -carbon is placed at the position susceptible to hydroxylation, resulting in the altered regioselectivity observed in the HO mutants.; To probe the mechanism of meso-carbon hydroxylation, azide complexes of pa-HO and nm-HO, as mimics of the ferric hydroperoxide intermediate, have been studied by NMR. A novel electronic structure with S = 3/2, (dxy,d yz)3(dxy) 1(dz2)1, companied with deformed heme, has been observed to exist in an equilibrium with S = 1/2, (dxy)2(d xy,dyz)3 electronic configuration and the equilibrium shifts to the former at an elevated temperature. These findings, together with the previous observations with pa-HO hydroxide complex (JACS 2003, 125, 11842), suggest that the microenvironment of the distal pocket in HO is unique in that it modulates the sigma-donating ability of the distal ligand through a hydrogen bond with the coordinated ligand. Consequently, the lowered ligand field strength promotes unique electronic structures and the deformation of heme that activate heme macrocycle to facilitate the hydroxylation of the meso-carbon susceptible to attack.; Heme oxygenation is an electron-dependent reaction, but the physiological electron donor of bacterial HO has not been identified. In this research, we characterized the protein encoded by bfd gene in P. aeruginosa to be a [2Fe-2S] ferredoxin and provided the first evidence suggesting pa-Bfd to be the physiological partner of pa-HO. The in vitro experiment results show that pa-Bfd participates in electron transfer from NADPH to pa-HO, which subsequently oxidizes ferric-heme to iron-biliverdin. |
