Heme oxygenase catalyzes the oxidative cleavage of protoheme to biliverdin, the first step of heme metabolism utilizing O 2 and NADPH. We determined the crystal structures of rat heme oxygenase-1 (HO-1)^heme and selenomethionyl HO-1^heme complexes. Heme is sandwiched between two helices with the N N-meso edge of the heme being exposed to the surface. Gly143N forms a hydrogen bond to the distal ligand of heme, OH 3 . The distance between Gly143N and the ligand is shorter than that in the human HO-1^heme complex. This difference may be related to a pH-dependent change of the distal ligand of heme. Flexibility of the distal helix may control the stability of the coordination of the distal ligand to heme iron. The possible role of Gly143 in the heme oxygenase reaction is discussed.z 2000 Federation of European Biochemical Societies.

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Heme oxygenase (HO) catalyzes physiological heme degradation consisting of three sequential oxidation steps that use dioxygen molecules and reducing equivalents. We determined the crystal structure of rat HO-1 in complex with heme and azide (HO-heme-N 3 ؊ ) at 1.9-Å resolution. The azide, whose terminal nitrogen atom is coordinated to the ferric heme iron, is situated nearly parallel to the heme plane, and its other end is directed toward the ␣-meso position of the heme. Based on resonance Raman spectroscopic analysis of HO-heme bound to dioxygen, this parallel coordination mode suggests that the azide is an analog of dioxygen. The azide is surrounded by residues of the distal F-helix with only the direction to the ␣-meso carbon being open. This indicates that regiospecific oxygenation of the heme is primarily caused by the steric constraint between the dioxygen bound to heme and the F-helix. The azide interacts with Asp-140, Arg-136, and Thr-135 through a hydrogen bond network involving five water molecules on the distal side of the heme. This network, also present in HO-heme, may function in dioxygen activation in the first hydroxylation step. From the orientation of azide in HO-heme-N 3 ؊ , the dioxygen or hydroperoxide bound to HO-heme, the active oxygen species of the first reaction, is inferred to have a similar orientation suitable for a direct attack on the ␣-meso carbon.

Heme oxygenase (HO) catalyzes the oxidative cleavage of heme to biliverdin by utilizing O(2) and NADPH. HO (apoHO) was crystallized as twinned P3(2) with three molecules per asymmetric unit, and its crystal structure was determined at 2.55 A resolution. Structural comparison of apoHO and its complex with heme (HO-heme) showed three distinct differences. First, the A helix of the eight alpha-helices (A-H) in HO-heme, which includes the proximal ligand of heme (His25), is invisible in apoHO. In addition, the B helix, a portion of which builds the heme pocket, is shifted toward the heme pocket in apoHO. Second, Gln38 is shifted toward the position where the alpha-meso carbon of heme is located in HO-heme. Nepsilon of Gln38 is hydrogen-bonded to the carbonyl group of Glu29 located at the C-terminal side of the A helix in HO-heme, indicative that this hydrogen bond restrains the angle between the A and B helices in HO-heme. Third, the amide group of Gly143 in the F helix is directed outward from the heme pocket in apoHO, whereas it is directed toward the distal ligand of heme in HO-heme. This means that the F helix around Gly143 must change its conformation to accommodate heme binding. The apoHO structure has the characteristic that the helix on one side of the heme pocket fluctuates, whereas the rest of the structure is similar to that of HO-heme, as observed in such hemoproteins as myoglobin and cytochromes b(5) and b(562). These structural features of apoHO suggest that the orientation of the proximal helix and the position of His25 are fixed upon heme binding.

Heme oxygenase (HO), 1 a microsomal enzyme that oxidizes protoheme to biliverdin IX␣, plays a key role in physiological heme metabolism. As shown in Scheme 1, the first step in the heme degradation catalyzed by HO is the oxidation of heme to ␣-hydroxyheme, a process requiring O 2 and reducing equivalents from NADPH-cytochrome P450 reductase (1). The second step is the formation of verdoheme with the concomitant release of the hydroxylated ␣-meso-carbon as CO (2, 3). This step is also O 2 -dependent. In the third step, biliverdin is formed from verdoheme in a reaction that again requires O 2 and reducing equivalents from NADPH-cytochrome P450 reductase (1).Our early stoichiometric study employing a reconstituted HO system suggested that 3 mol of O 2 and 4 -5 mol of NADPH were consumed during the degradation of 1 mol of heme by HO (4 Recently we have established a system for expressing and purifying a truncated version of the inducible rat enzyme (HO-1) consisting of Met-1 through Pro-267 (rHO-1); the sequence (1-267) of this truncated protein is completely identical to the corresponding sequence of the rat HO-1 (7). About 130 mg of soluble and catalytically active rHO-1 can be routinely obtained from a 10-liter culture (8). In an attempt to resolve the discrepancy arising from the work of Matera et al. (5) and Liu et al. (6), we have prepared ferric ␣-hydroxyheme, complexed it with purified rHO-1, and characterized its oxidation to verdoheme under various conditions by optical and ESR spectroscopies. EXPERIMENTAL PROCEDURESMaterials-rHO-1 lacking the 22-amino acid C-terminal hydrophobic stretch was expressed in Escherichia coli and purified as described (8). The specific activity of rHO-1, 8300 units/mg protein (8), slightly exceeded that of native HO-1 from rat liver, 4500 -6000 units/mg protein (9), even taking into account the reduction in molecular mass by truncation. In addition, rHO-1, when complexed with hemin, catalyzed the heme degradation reaction to biliverdin in a manner spectrophotometrically the same as native HO-1 (data not shown). The purification of NADPH-cytochrome P450 reductase from rat liver was accomplished using DEAE-Sephadex and 2Ј,5Ј-ADP-Sepharose (10) column chromatographies. Gases of high purity, argon (99.999%), O 2 (99.99%), and N 2 (99.99%) were obtained from Iwatani, Fukuoka, Japan. All chemicals used were of analytical grade and were obtained commercially.Preparation of ␣-Hydroxyheme-rHO-1 Complex and Titration of the ␣-Hydroxyheme-rHO-1 Complex with O 2 -␣-Benzoyloxyprotoporphyrin IX dimethyl ester was synthesized according to the method of Sano et al. (11), and its regiochemical structure was confirmed by a one-and two-dimensional 1 H NMR (300 MHz) spectra with a Bruker DSX 300 spectrometer. Insertion of iron was performed as described (12) to obtain ␣-benzoyloxyprotohemin dimethyl ester. The amount of ␣-hydroxyhemin dimethyl ester was determined as pyridine hemochrome using ⑀ 422 nm ϭ 153.6 mM Ϫ1 cm Ϫ1 in CHCl 3 (11). Alkaline hydrolysis of ␣-benzoyloxyprotohemin dimethyl este...