The Crystal Structures of the Ferric and Ferrous Forms of the Heme Complex of HmuO, a Heme Oxygenase of Corynebacterium diphtheriae

Abstract

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 ...

Application of the paramagnetic dipole field for solution NMR active site structure determination in low‐spin, cyanide‐inhibited ferric hemoproteins

“…Indeed, 1 H 2D NMR characterization of human (16, 43) and a bacterial (17) cyanide-inhibited HO-substrate complex, with 1 H NMR spectra illustrated in Figure 16, together with the crystal coordinates for the homologous aquo complexes (42,44); yielded magnetic axes (16) with excellent correlation between d dip (calc), and d dip (obs), as illustrated (16) for the human HO-protohem-CN complex in Figure 17 (36). Adapted from (38,39), with permission.…”

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

“…Although most of the structural and functional studies have been conducted on the soluble, truncated form of isoform-1 of mammalian heme oxygenase, HO-1 (6 -8), the enzyme is also present in some pathogenic bacteria where it is essential for heme-based iron acquisition from a host lacking in free extracellular iron (9 -11). Two HO proteins from pathogenic bacteria have been characterized in some detail, namely HmuO from Corynebacterium diphtheriae and HemO from Neisseria meningitidis (12)(13)(14)(15)(16). In comparison with the mammalian HO, neither of them is membrane-bound.…”

“…HmuO has 33% sequence identity to the first 221 amino acids of human HO-1. Despite their differences in size, the two bacterial HO proteins have overall protein folds, heme environments, and catalytic mechanisms very similar to those for mammalian HO-1 (12)(13)(14)(15)(16).…”

“…Analysis of the Fe-O 2 vibration modes predicts that the Fe-O-O unit is highly bent by steric interactions between the bound O 2 and the residues in the distal pocket (20). 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).…”

“…However, the structure of the oxy complex is needed to more accurately delineate the mode of oxygen binding. The heme oxygenase from C. diphtheriae (HmuO) has been thoroughly characterized structurally and enzymatically (12)(13)(14), and we have now been able to obtain crystals of the oxy form of this enzyme. Oxygenated crystals were obtained by using strictly anaerobic conditions for reduction of ferric crystals, carefully removing excess reducing agent, exposing the one-electron reduced sample to mother liquor equilibrated with 1 atm of O 2 for 10 min, and then flash freezing the final oxygenated samples.…”