Tài liệu Báo cáo khoa học: Unique features of recombinant heme oxygenase of Drosophila melanogaster

Unique features of recombinant heme oxygenase of Drosophila

melanogaster compared with those of other heme

oxygenases studied

Xuhong Zhang1

, Michihiko Sato2

, Masanao Sasahara1

, Catharina T. Migita3 and Tadashi Yoshida1

1

Department of Biochemistry and 2

Central Laboratory for Research and Education, Yamagata University School of Medicine, Japan; 3

Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan

We cloned a cDNA for a Drosophila melanogaster homo￾logue 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 hexa￾coordinated 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 oxy￾genase; 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-stepoxidation 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 micro￾somal 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

[12–14] and plants [15–18] 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 photo￾responsive bilins such as phycobilins and phytochromobi￾lins [15–19]. Although the HOs have been characterized

structurally and functionally in most species, very little is

known about HO in insects.

Heme is extremely important in insects. It is a vital

nutrient for most, if not all, insects for their embryonic

development [20], although they do not use it as a transport

vehicle or storage vessel for oxygen. Heme also serves as the

prosthetic moiety of hemoproteins, such as hemoglobin

[21,22], catalase [23] and nitric oxide synthase [24], which are

essential for biological function. However, heme is poten￾tially toxic to insects, particularly blood-sucking insects such

as mosquitoes, because it catalyzes oxidative reactions that

can damage membranes and destroy nucleic acids. There￾fore, insects are thought to have several mechanisms for

sequestering and controlling heme. For example, it can be

conjugated with such proteins as the heme binding protein

Correspondence to T. Yoshida, Department of Biochemistry,

Yamagata University School of Medicine, Yamagata, Japan.

Fax: + 81 23 628 5225, Tel.: + 81 23 628 5222,

E-mail: [email protected]

Abbreviations: HO, heme oxygenase; CPR, NADPH/cytochrome

P450 reductase; DmHO, heme oxygenase of D. melanogaster;

DmDHO, truncated form of D. melanogaster heme oxygenase;

DmCPR, NADPH/cytochrome P450 reductase of D. melanogaster;

DmDCPR, truncated form of D. melanogaster NADPH/cytochrome

P450 reductase; Syn HO-1, heme oxygenase-1 of Synechocistis sp.

PCC 6803.

Enzymes: heme oxygenase (EC 1.14.99.3); NADPH/cytochrome P450

reductase (EC 1.6.2.4).

(Received 25 December 2003, revised 2 March 2004,

accep ted 9 March 2004)

Eur. J. Biochem. 271, 1713–1724 (2004) FEBS 2004 doi:10.1111/j.1432-1033.2004.04077.x