Ferric α-Hydroxyheme Bound to Heme Oxygenase Can Be Converted to Verdoheme by Dioxygen in the Absence of Added Reducing Equivalents

Abstract

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...

Electrochemical reduction of ferrous α-verdoheme in complex with heme oxygenase-1

“…1 [4,5]. The first step is the oxidation of heme to α-hydroxyheme, requiring O 2 and two electrons [6][7][8]. The second step, also requiring O 2 and an electron, is the formation of ferrous α-verdoheme from α-hydroxyheme with the concomitant release of hydroxylated α-meso carbon as CO [9][10][11][12].…”

Crystal Structure of Rat Heme Oxygenase-1 in Complex with Heme Bound to Azide

“…The second step is conversion of ␣-hydroxyheme to verdoheme with the concomitant release of the ␣-meso carbon as CO (10). Whether or not the ferric ␣-hydroxyheme in complex with HO requires an additional electron before reacting with dioxygen to yield verdoheme remains controversial (11)(12)(13)(14). Lastly, the oxygen bridge of verdoheme is cleaved, producing iron and biliverdin.…”

Hydroxylamine and hydrazine bind directly to the heme iron of the heme–heme oxygenase-1 complex

“…The amine-bound forms of the ferric heme-rHO-1 complex were prepared as follows: a portion of the concentrated complex solution was transferred to a custom-made anaerobic titrator [4] with a 2-mm path length cuvette, in which $1 ml of 0.1 M potassium phosphate buffer (pH 7.4) had been placed and the gas phase had been replaced with argon. The final concentration of the complex was 37 lM.…”

“…The HO reaction consists of three steps in which two distinct intermediates, namely a-hydroxyheme [3][4][5] and verdoheme [6,7], are formed. The first step, the a-meso carbon hydroxylation of heme to a-hydroxyheme, is intriguing because of its unique mechanism of oxygen activation.…”