We cloned a cDNA for a Drosophila melanogaster homologue of mammalian heme oxygenase (HO) and constructed a bacterial expression system of a truncated, soluble form of D. melanogaster HO (DmDHO). The purified DmDHO degraded hemin to biliverdin, CO and iron in the presence of reducing systems such as NADPH/cytochrome P450 reductase and sodium ascorbate, although the reaction rate was slower than that of mammalian HOs. Some properties of DmHO, however, are quite different from other known HOs. Thus DmDHO bound hemin stoichiometrically to form a hemin-enzyme complex like other HOs, but this complex did not show an absorption spectrum of hexacoordinated heme protein. The absorption spectrum of the ferric complex was not influenced by changing the pH of the solution. Interestingly, an EPR study revealed that the iron of heme was not involved in binding heme to the enzyme. Hydrogen peroxide failed to convert it into verdoheme. A spectrum of the ferrous-CO form of verdoheme was not detected during the reaction from hemin under oxygen and CO. Degradation of hemin catalyzed by DmDHO yielded three isomers of biliverdin, of which biliverdin IXa and two other isomers (IXb and IXd) accounted for 75% and 25%, respectively. Taken together, we conclude that, although DmHO acts as a real HO in D. melanogaster, its active-site structure is quite different from those of other known HOs.Keywords: biliverdin; Drosophila melanogaster; heme oxygenase; insect; NADPH/cytochrome P450 reductase.Heme oxygenase (HO, EC 1.14.99.3) was first characterized in mammals as a microsomal enzyme that catalyzes the three-step oxidation of hemin to biliverdin IXa, CO, and free iron, via a-meso-hydroxyhemin, verdoheme, and ferric iron-biliverdin complex [1-3] (Scheme 1). To date two mammalian isozymes of HO have been identified [4]: HO-1, an inducible enzyme that is highly expressed in the spleen and liver; HO-2, a constitutive enzyme found abundantly in the brain and testes. The two isozymes have about 43% similarity at amino acid level, and both have a C-terminal hydrophobic domain that is involved in binding to microsomal membrane. Both HO-1 and HO-2 have been demonstrated to play important roles in physiological iron homeostasis [5,6], antioxidant defense [7,8], and possibly the cGMP signaling pathway [9,10]. Although HO-3 was once reported as an isozyme of HO, its function is not yet well defined [11].HO has also been found and characterized in bacteria [12][13][14] and plants [15][16][17][18] and other species such as Rhodophyta [19]. In contrast with mammalian HO, these HOs are water-soluble enzymes because they lack a membrane-anchoring domain at the C-termini of their sequences. In pathogenic bacteria, HO is thought to help bacteria to acquire iron from heme-containing proteins found in their host cells for survival and toxin production. In plants, biliverdin is used for the biosynthesis of photoresponsive bilins such as phycobilins and phytochromobilins [15][16][17][18][19]. Although the HOs have been characterized structurally and functional...

The hemin complex of Hmu O, a 24-kDa soluble heme degradation enzyme in Corynebacterium diphtheriae, is coordinated axially to a neutral imidazole of a proximal histidine residue in Hmu O. To identify which of the eight histidines in Hmu O is the proximal heme ligand, we have constructed and expressed the plasmids for eight His 3 Ala Hmu O mutants. Reconstituted with hemin, the active site structures and enzymatic activity of these mutants have been examined by EPR, resonance Raman, and optical absorption spectroscopy.

Two isoforms of a heme oxygenase gene, ho1 and ho2, with 51% identity in amino acid sequence have been identified in the cyanobacterium Synechocystis sp. PCC 6803. Isoform‐1, Syn HO‐1, has been characterized, while isoform‐2, Syn HO‐2, has not. In this study, a full‐length ho2 gene was cloned using synthetic DNA and Syn HO‐2 was demonstrated to be highly expressed in Escherichia coli as a soluble, catalytically active protein. Like Syn HO‐1, the purified Syn HO‐2 bound hemin stoichiometrically to form a heme–enzyme complex and degraded heme to biliverdin IXα, CO and iron in the presence of reducing systems such as NADPH/ferredoxin reductase/ferredoxin and sodium ascorbate. The activity of Syn HO‐2 was found to be comparable to that of Syn HO‐1 by measuring the amount of bilirubin formed. In the reaction with hydrogen peroxide, Syn HO‐2 converted heme to verdoheme. This shows that during the conversion of hemin to α‐meso‐hydroxyhemin, hydroperoxo species is the activated oxygen species as in other heme oxygenase reactions. The absorption spectrum of the hemin–Syn HO‐2 complex at neutral pH showed a Soret band at 412 nm and two peaks at 540 nm and 575 nm, features observed in the hemin‐Syn HO‐1 complex at alkaline pH, suggesting that the major species of iron(III) heme iron at neutral pH is a hexa‐coordinate low spin species. Electron paramagnetic resonance (EPR) revealed that the iron(III) complex was in dynamic equilibrium between low spin and high spin states, which might be caused by the hydrogen bonding interaction between the distal water ligand and distal helix components. These observations suggest that the structure of the heme pocket of the Syn HO‐2 is different from that of Syn HO‐1.