Unique features of recombinant heme oxygenase of Drosophila melanogaster compared with those of other heme oxygenases studied

Abstract

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...

A heme-degradation pathway in a blood-sucking insect

“…Recently, two exceptions were reported: the Pseudomonas aeruginosa HO, capable of degrading heme to both ␦ and ␤ isomers of BV (25), and the recombinant Drosophila melanogaster HO, which produced simultaneously ␦, ␤, and ␣ BV isomers in vitro (26). Because it is well established that some insects produce BV IX␥ (18), we investigated the possibility that RpBV is also the ␥ isomer.…”

Retrograde bilin signaling enables Chlamydomonas greening and phototrophic survival

“…1B), consistent with high-throughput mammalian, cyanobacterial, and plant heme oxygenases that are coupled with an NADPH-dependent BV reductase (BVR) or with a ferredoxin-dependent bilin reductase (FDBR) such as PCYA or HY2 (31,32). Heme oxygenases with relaxed regiospecificity similar to that of HMOX2 have been documented from insects, organisms that lack light-harvesting biliproteins, oxygen-carrying hemoproteins, and α-specific BV reductases (34). Recombinant PCYA was also active, with the core FDBR region of PCYA proving sufficient for conversion of BV IXα to PCB (Fig.…”

The Heme Oxygenase(s)-Phytochrome System of Pseudomonas aeruginosa

“…BphO Prefers Ferredoxins and Ascorbate as Reducing Partners-Most HOs are capable of utilizing an endogenous E. coli electron donor, which is manifested as a green color in E. coli cells expressing HOs (3,27,43). In vitro, however, the activity of most bacterial HOs is usually tested using either mammalian CPR or ascorbate as an electron donor.…”