Crystal structures of the ferric and ferrous heme complexes of HmuO, a 24-kDa heme oxygenase of Corynebacterium diphtheriae, have been refined to 1.4 and 1.5 Å resolution, respectively. The HmuO structures show that the heme group is closely sandwiched between the proximal and distal helices. The imidazole group of His-20 is the proximal heme ligand, which closely eclipses the ␤-and ␦-meso axis of the porphyrin ring. A long range hydrogen bonding network is present, connecting the iron-bound water ligand to the solvent water molecule. This enables proton transfer from the solvent to the catalytic site, where the oxygen activation occurs. In comparison to the ferric complex, the proximal and distal helices move closer to the heme plane in the ferrous complex. Together with the kinked distal helix, this movement leaves only the ␣-meso carbon atom accessible to the iron-bound dioxygen. The heme pocket architecture is responsible for stabilization of the ferric hydroperoxo-active intermediate by preventing premature heterolytic O-O bond cleavage. This allows the enzyme to oxygenate selectively at the ␣-meso carbon in HmuO catalysis.Biological heme catabolism is conducted by a family of enzymes termed as heme oxygenase (HO), 1 which catalyzes oxidative degradation of iron protoporphyrin IX (heme hereafter) to biliverdin IX, iron, and CO in the presence of reducing equivalents (1). In mammalian systems where electrons are supplied by NADPH through NADPH-cytochrome P450 reductase (2), HO is the enzyme responsible for excess heme excretion and iron recycling (3). The product CO has been implicated as a messenger molecule in various physiological functions (4 -6). In pathogenic bacteria, HO is essential for heme-based iron acquisition from a host lacking in free extracellular iron (7-9). Major advances have been made in understanding the structure and function of HO by using catalytically active, truncated, water-soluble forms of recombinant HO-1, an inducible isoform of mammalian HO (10 -13). HO is not a hemeprotein by itself but utilizes heme as both a prosthetic group and a substrate. In its catalytic cycle, HO first binds 1 eq of heme to form a ferric heme-HO complex (Fig. 1). The first electron donated from the reducing equivalent reduces the heme iron to the ferrous state. Then O 2 binds to it to form a meta-stable oxy complex. One-electron reduction of the oxy form generates ferric hydroperoxo, which self-hydroxylates the ␣-meso carbon of the porphyrin ring to form the ferric ␣-meso-hydroxyheme intermediate (12,13). This is different from P450 enzymes, in which the O-O bond of the hydroperoxo is heterolytically cleaved to generate a ferryl (Fe 4ϩ ϭO) hydroxylating active intermediate (14). Ferric ␣-meso-hydroxyheme in HO exits as a ferric oxopholin resonance structure that includes a ferrous porphyrin neutral radical (11). Upon reaction with O 2 and one electron, ferric ␣-meso-hydroxyheme is converted to ferrous verdoheme. This conversion has been proposed to be initiated by the dioxygen reaction with the ferrous ...

HmuO, a heme oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme. The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography. The oxygen association and dissociation rate constants are 5 M ؊1 s ؊1 and 0.22 s ؊1 , respectively, yielding an O 2 affinity of 21 M ؊1 , which is ϳ20 times greater than that of mammalian myoglobins. However, the affinity of HmuO for CO is only 3-4-fold greater than that for mammalian myoglobins, implying the presence of strong hydrogen bonding interactions in the distal pocket of

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