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 ...

Heme-binding protein 23 kDa (HBP23), a rat isoform of human proliferation-associated gene product (PAG), is a member of the peroxiredoxin family of peroxidases, having two conserved cysteine residues. Recent biochemical studies have shown that HBP23/ PAG is an oxidative stress-induced and proliferation-coupled multifunctional protein that exhibits specific bindings to c-Abl protein tyrosine kinase and heme, as well as a peroxidase activity. A 2.6-Å resolution crystal structure of rat HBP23 in oxidized form revealed an unusual dimer structure in which the active residue Cys-52 forms a disulfide bond with conserved Cys-173 from another subunit by C-terminal tail swapping. The active site is largely hydrophobic with partially exposed Cys-173, suggesting a reduction mechanism of oxidized HBP23 by thioredoxin. Thus, the unusual cysteine disulfide bond is involved in peroxidation catalysis by using thioredoxin as the source of reducing equivalents. The structure also provides a clue to possible interaction surfaces for c-Abl and heme. Several significant structural differences have been found from a 1-Cys peroxiredoxin, ORF6, which lacks the C-terminal conserved cysteine corresponding to Cys-173 of HBP23.

Bitiscetin, a C-type lectin-like protein isolated from the venom of the snake Bitis arientans, promotes the interactions between plasma von Willebrand factor (VWF) and platelet membrane glycoprotein Ib (GPIb) to induce platelet aggregation. We report here the crystal structure of bitiscetin at 2.0 A resolution. The overall fold is similar to those of coagulation factor IX/X-binding protein (IX/X-bp) and flavocetin-A (a GPIb-binding protein), although these three proteins are functionally distinct from one another. The characteristic property determining target recognition is explained mainly by the differences in the surface potential on the central concave surface. A negatively charged patch on the surface of bitiscetin is a candidate for the site of binding to the positively charged surface of the VWF A1 domain, as shown in the case of another platelet aggregation inducer, botrocetin. However, a positively charged patch near the central concave surface is unique for bitiscetin and suggests that it is the binding site for the negatively charged surface of the VWF A3 domain. Thus, the interactions accounting for VWF activation by bitiscetin possibly involve both the A1 and A3 domains of VWF, indicating a specific mechanism of VWF activation by bitiscetin.

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