Rat liver heme oxygenase. High level expression of a truncated soluble form and nature of the meso-hydroxylating species.

A rat heme oxygenase (HO-1) gene without the sequence coding for the last 23 amino acids has been constructed and expressed behind the pho A promoter in Escherichia coli. The enzyme is expressed at high levels as a soluble catalytically active protein that causes the bacterial cells to accumulate biliverdin. The purified truncated heme-heme oxygenase complex is spectroscopically indistinguishable from the complex with the native enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P450 reductase. Reaction of the recombinant heme-heme oxygenase complex with H2O2 produces a species with the spectroscopic properties of verdoheme. Unidentified products are obtained when this intermediate is directly extracted from the protein, but biliverdin is obtained if the verdoheme-protein complex is exposed to cytochrome P450 reductase and NADPH before the extraction step. In contrast, reaction of the heme-heme oxygenase complex with meta-chloroperbenzoic acid (mCPBA), tert-butylhydroperoxide, or cumene hydroperoxide yields a ferryl (FeIV = O) complex. Reaction of the heme-heme oxygenase complex with mCPBA also produces an EPR-detectable protein radical. In accord with formation of a ferryl intermediate, recombinant heme oxygenase catalyzes the mCPBA- and alkylhydroperoxide-dependent peroxidation of 2-methoxyphenol (guaiacol). Guaiacol oxidation is not observed during turnover of the enzyme by cytochrome P450 reductase/NADPH or H2O2. Conversely, biliverdin is not formed with tert-butylhydroperoxide or mCPBA. H2O2 thus supports the first step of the normal catalytic oxidation of heme by heme oxygenase, but alkyl and acyl hydroperoxides do not. These results suggest that the alpha-meso-hydroxylation required for biliverdin formation is mediated by the distal of the two oxygens in the iron-dioxygen intermediate (Fe-O-O) engendered by reaction with either cytochrome P450 reductase/NADPH or H2O2.

From the DeDartment of Pharmaceutical Chemistrv. School of Pharmacy, and Liver Center, University of California, Sun Francisco, California 94143-0446 ", A rat heme oxygenase (HO-1) gene without the sequence coding for the last 23 amino acids has been constructed and expressed behind the pho A promoter in Escherichia coli. The enzyme is expressed at high levels as a soluble catalytically active protein that causes the bacterial cells to accumulate biliverdin. The purified truncated heme-heme oxygenase complex is spectroscopically indistinguishable from the complex with the native enzyme and converts heme to biliverdin when reconstituted with rat liver cytochrome P460 reductase. Reaction of the recombinant heme-heme oxygenase complex with H202 produces a species with the spectroscopic properties of verdoheme. Unidentified products are obtained when this intermediate is directly extracted from the protein, but biliverdin is obtained if the verdoheme-protein complex is exposed to cytochrome P460 reductase and NADPH before the extraction step. In contrast, reaction of the heme-heme oxygenase complex with meta-chloroperbenzoic acid (mCPBA), tert-butylhydroperoxide, or cumene hydroperoxide yields a ferryl (Ferv=O) complex. Reaction of the heme-heme oxygenase complex with mCPBA also produces an EPRdetectable protein radical. In accord with formation of a ferryl intermediate, recombinant heme oxygenase catalyzes the mCPBA-and alkylhydroperoxide-dependent peroxidation of 2-methoxyphenol (guaiacol). Guaiacol oxidation is not observed during turnover of the enzyme by cytochrome P460 reductaseiNADPH or H202. Conversely, biliverdin is not formed with tert-butylhydroperoxide or mCPBA. Hz02 thus supports the first step of the normal catalytic oxidation of heme by heme oxygenase, but alkyl and acyl hydroperoxides do not. These results suggest that the a-meso-hydroxylation required for biliverdin formation is mediated by the distal of the two oxygens in the iron-dioxygen intermediate (Fe-0-0) engendered by reaction with either cytochrome P460 reductaseNADPH or H20a.
Heme oxygenase catalyzes the NADPH-and cytochrome P450 reductase-dependent oxidation of heme' to biliverdin (1). * This work was supported by Grants DK30297 and GM32488 from the National Institutes of Health. The University of California San Francisco Liver Center spectrophotometry and mass spectrometry facilities used in this work were supported by National Institutes of Health Grant 5 P30 DK26743. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
School of Pharmacy, University of California, San Francisco, CA 94143-$ To whom correspondence and reprint requests should be addressed:

0446.
less of the oxidation and ligation state of the iron; HPLC, high pressure The abbreviations used are: heme, iron protoporphyrin M regardliquid chromatography; FPLC, fast protein liquid chromatography; PAGE, polyacrylamide gel electrophoresis; mCPBA, metu-chloroperben-The enzyme is a membrane-bound protein that has been purified from several sources, including rat liver (21, pig spleen (31, bovine spleen (41, and chicken liver (5). The cDNAs for the rat, human, and chicken enzymes have been isolated and sequenced (6)(7)(8). l b o forms of the enzyme, known as HO-1 and HO-2, have so far been identified, although most of the data available in the literature is on the HO-1 form (9, 10). The predicted molecular mass for the rat enzyme is 33.0 kDa, in close agreement with the experimental value (2). Both the rat and human enzymes have hydrophobic segments of 22 amino acids at the carboxyl terminus that are important for their insertion into the endoplasmic reticulum (6,7). It has been shown that heme oxygenase synthesized in a cell-free system is post-translationally integrated into the microsomal membrane (11). Treatment of the membrane-bound protein with trypsin results in release of a 28-kDa domain that is still able to bind heme and to form the ferric, ferrous, ferrous-CO and ferrous-02 complexes, although the ability of the complex to interact with cytochrome P450 reductase is impaired (11,12). Partial proteolytic digestion of the full-length heme oxygenase expressed in E. coli produces a variety of fragments, including a 30-kDa fragment that retains the ability to accept the second electron from cytochrome P450 reductase and catalyze biliverdin formation (13).
Heme oxygenase employs heme as both prosthetic group and substrate. Reduction of the enzyme-bound heme by cytochrome P450 reductase yields the ferrous form of the complex that readily binds oxygen or carbon monoxide (2). The catalytic cycle of heme oxygenase parallels that of cytochrome P450 in that the ferrous dioxygen complex accepts a second electron from cytochrome P450 reductase and is thereby transformed into an activated oxidizing species (14). Formation of this oxidizing species results in addition of a hydroxyl group to the a-methene bridge to give a-meso-hydroxyheme (15)(16)(17). The oxygen added to the meso-carbon derives from molecular oxygen (18). The nature of the oxidizing species is unknown, although model studies indicate that redox active metalloporphyrin complexes can react with HzOZ under appropriate conditions to give mesohydroxylated products (19). The a-meso-hydroxyheme is subsequently oxidized to carbon monoxide and an enzyme-bound intermediate with Am, = 688 nm. Binding of carbon monoxide to the iron of the enzyme-bound intermediate shifts the absorption maximum to 638 nm (20). The intermediate is thought to be verdoheme (Scheme l), although its structure has not been unambiguously established (21,22). 14C labeling studies have clearly demonstrated, however, that the methene bridge carbon is eliminated as carbon monoxide (23). The final step of the catalytic process, which requires both molecular oxygen and additional electrons from NADPH via cytochrome P450 reductase, produces biliverdin M a from the verdoheme-like intermezoic acid; guaiacol, 2-methoxyphenol. diate (24). The oxygens in the carbon monoxide and the two lactam carbonyls of biliverdin derive from molecular oxygen (18). Indeed, studies with mixtures of lSOz and lSOz indicate that the two lactam carbonyl oxygens derive from different oxygen molecules (251, in accord with a mechanism in which one of the oxygens is introduced during the formation of verdoheme and the other in the conversion of verdoheme to biliverdin (Scheme 1).
We report here construction of a gene coding for a truncated version of rat heme oxygenase HO-1, development of a highyield expression system that produces the soluble, fully active truncated enzyme, and highly informative differences in the reactions of the recombinant enzyme with HzOz and alkylhydroperoxides.

EXPERIMENTAL PROCEDURES
General Methods-Plasmid purification, subcloning, and bacterial transformations were carried out as described (26). Deionized doubly distilled water was used for all biochemical experiments. Oligonucleotides were synthesized at the University of California, San Francisco, Biomolecular Resource Center using an Applied Biosystems 380B DNA synthesizer. The oligonucleotides were further purified on mini-spin columns (Worthington). HPLC was done on a Hewlett-Packard Series I1 1090 Liquid Chromatograph.
Construction of the pBAH030 Vector-The expression vector containing the heme oxygenase (HO-1) gene was constructed in pBAce (28). A pKK233-2 vector constructed as described by Ishikawa et al. (13) from the full-length cDNA pRHOl provided by Shibahara et al. (6) in the Okayama-Berg vector was used to obtain by polymerase chain reaction the truncated gene for cloning into pBAce. The 5"sense oligonucleotide (5'-GAAACAGCATATGGAGCGCCCACAG-3') coded for an N&I site at the NH, terminus. The 3"antisense oligonucleotide (5"CCAACAGTC-GACTI'ACATGGCATAA-3') encoded the termination codon TAA at the proline 23 amino acids from the COOH terminus and immediately following the TAA codon at a SalI site. The reaction conditions were as follows: 94 "C for 7 min, followed by 94 "C for 1 min, 40 "C for 1 min, 72 "C for 2 min, with the amplification repeated for five cycles. The reaction was then continued at 94 "C for 1 min, 60 "C for 1 min, 72 "C for 2 min for 30 cycles, and finally at 72 "C for 10 min. The target fragment was gel-purified and digested with NdeI and SalI and cloned into pBAce (Fig. 1). Transformants were screened by restriction digestion and confirmed by sequencing.
Expression and Purification of the Duncated Heme Oxygenase-A with fresh colonies of transformed E. coli DH5aF'. From the fresh 3-ml inoculum in low phosphate induction media was set up from plates mid-log phase cultures 100 pl was used to inoculate 1-liter cultures of the same media. The cells were grown at 30 "C for 18 h or until the media became green. Expression of the protein for periods over 24 h results in partial proteolysis of the 30-kDa form to a 28-kDa protein.
The harvested cells were lysed in 50 m~ Tris buffer (pH 8.0) containing 6 m~ MgClz, 1 m~ dithiothreitol, 1 m~ EDTA, and 1 m~ phenylmethylsulfonyl fluoride in a Bio-Spec Bead Beater. The cells were then spun at 27,000 x g for 60 min. Ammonium sulfate was added to the resulting supernatant to a final concentration of 30% of saturation and the solution was stirred for 60 min. Following centrifugation (27,000 x g for 20 mid, the ammonium sulfate concentration was raised to 60% of saturation. The 3040% pellets were collected and resuspended in 10 m~ potassium phosphate buffer (pH 7.4) (buffer A). The protein was applied to a Mono Q (HR 10/10) column, and the protein was eluted with a step gradient of 10 m~ potassium phosphate (pH 7.4) containing 250 m~ KC1 (buffer B). The gradient was increased linearly from 0 to 25% buffer B, held for 10 min at 25% buffer B, and then increased linearly to 100% buffer B. The fractions in the first half of the gradient containing heme oxygenase activity were pooled and dialyzed against buffer A.
Spectroscopic Assay of Heme Oxygenase Actiuity-Heme oxygenase activity was assayed as previously described (2). The assays contained heme oxygenase (3 pg, 0.1 nmol), 15 p~ hemin, 1 p~ bovine serum albumin, an excess of partially purified biliverdin reductase (rat liver cytosol), and purified cytochrome P450 reductase (0.3 nmol) in a final volume of 1 ml of 100 m~ potassium phosphate buffer (pH 7.4). Cytochrome P450 reductase was purified as reported by Yasukochi and Masters (29). The reaction was initiated by adding NADPH to a final concentration of 100 p~. The rate of bilirubin formation at 37 ' C was monitored at 468 nm and calculated using an extinction coefficient of 43.5 m " l cm".
HPLC of the Heme Oxygenase Reaction Products-After reaction of the heme-heme oxygenase complex with cytochrome P450 reductase and NADPH, glacial acetic acid (150 pl) and 6 N HCI (200 pl) were added to the reaction (1 ml) before extracting it with chloroform (1 ml). The organic extract was washed with distilled water (3 x 1 ml), and the solvent was then removed under a stream of nitrogen (27). The residue was analyzed by reverse phase HPLC on a Partisil ODs-3 (5 pm, C18) column eluted with 0.1 M dioctylamine and 0.1 M acetic acid in methanol at a flow rate of 0.5 drnin., The eluent was monitored at 380 nm. Biliverdin M a elutes at 7.8 min and heme at 9.5 and 10.2 min. Heme presumably elutes as two peaks due to exchange of the hemin chloride ligand for a hydroxyl or other ion.
Purification of the Heme-Heme Oxygenase Complex-The heme-heme oxygenase complex was prepared essentially as described previously (2). Hemin was added to the FPLC-purified heme oxygenase to give a final 2:l heme:protein ratio. The sample was applied to a Bio-Gel HTP column (15 x 60 mm) pre-equilibrated with buffer A. The column was then washed in the same buffer until no heme could be detected in the eluent with a W monitor set at 402 nm. The heme-heme oxygenase complex was finally eluted with 110 m~ potassium phosphate buffer (pH 7.4). Protein purity was checked by SDS-PAGE on 12.5% polyacrylamide gels (30). In addition, a 20-pg sample was subjected to SDS-PAGE and transferred to a Pro-Blot membrane (Bio-Rad). The major band was removed from the membrane and subjected to NHz-terminal sequencing on an Applied Biosystems model 470A protein sequencer (Biomolecular Resource Center, University of California, San Francisco).
Absorption and EPR Spectroscopy-The spectra of the heme-heme oxygenase complex were recorded on a Hewlett-Packard 8450 A spectrophotometer. The reduced ferrous-CO complex was formed by addition of dithionite to a carbon monoxide-saturated solution of the ferric complex. The ferrous-0, complex was obtained, as described previously (2), by passing the carbon monoxide complex down a Sephadex G25 column pre-equilibrated with 10 m~ potassium phosphate (pH 7.4) buffer.
immediately after addition of 1-5 eq of mCPBA. EPR spectra of the The heme-heme oxygenase complex was frozen in liquid nitrogen frozen samples were recorded under the following conditions; gain, 4.0 x 104; microwave power, 2 milliwatts; modulation intensity, 8 G , time constant, 0.5 s; scan time, 4 min. The magnetic field was set at 3269 G, and the spectra were recorded over a 500-G range.
Reactions of Heme Oxygenase with Peroxides-One equivalent of 1 m~ H20n (10 pl) in 0.1 M potassium phosphate buffer (pH 7.4) was added to a solution of the heme-heme oxygenase complex (10 p~) in the same buffer. The reaction was monitored spectroscopically. Following maximum loss of the Soret band and maximum increase in the absorption at approximately 680 nm, pyridine was added to the solution to a final concentration of 20%. The product was extracted into chloroform (1 ml) and the solvent was removed under a stream of nitrogen. In a separate reaction, pyridine was added to half of the solution obtained from reaction of the heme-heme oxygenase complex with 1 eq of H202, whereas the remainder of the solution was allowed to react with cytochrome P450 reductase and NADPH as described below. The product formed in the reaction with cytochrome P450 reductase, and NADPH was extracted for analysis by UV and HPLC as described above.
Reaction of Heme Oxygenase with Cytochrome P450 Reductase and NADPH--Cytochrome P450 reductase (42 1.1~; 31, reductase:heme oxygenase) and NADPH (14 PM) were added to a cuvette containing a solution of the heme-heme oxygenase complex (14 p~) in 0.1 M potassium phosphate buffer (pH 7.4) that had been presaturated with carbon monoxide by bubbling with the gas. The progress of the reaction was monitored by UV spectroscopy. After approximately 10 min, a t which point no further change occurred in the spectrum, pyridine (20% final concentration) was added. The mixture was extracted with chloroform and the product isolated by removing the organic solvent under a stream of nitrogen. Under these conditions the reaction is arrested a t the verdoheme stage. In some experiments, the verdoheme intermediate accumulated in the presence of carbon monoxide was not extracted but was allowed to continue to the fully oxidized product by displacing the carbon monoxide with oxygen in the presence of NADPH before isolating the product for HPLC analysis.

RESULTS
Expression and Purification of Duncated Heme Oxygenase -A truncated heme oxygenase gene without the bases coding for the terminal 23 amino acids was constructed in pBAce ( Fig.  1) and expressed in E. coli. The soluble truncated form of the protein thus obtained in excellent yield was fully active (see below). Expression of the truncated heme oxygenase gene turns the medium green due to the accumulation of biliverdin, as reported earlier for expression of the full-length gene (13). The bacterial cells therefore have a reductase activity that supports the catalytic turnover of heme oxygenase.   -, ferric complex and cytochrome P450 reductase; . . . ., immediately following the addition of NADPH; 10 min after addition of NADPH.
Purification of the truncated heme-heme oxygenase complex by ammonium sulfate fractionation and FPLC (Table  I) yielded a protein that gave a single band at 30 kDa on SDS-PAGE (Fig. 2). NH2-terminal sequencing of the 30-kDa band showed that the first 5 amino acids (MERPQ) were identical to those of the rat liver enzyme. The amount of protein purified from 3 liters of cells was estimated to be 104 mg, based on an extinction coefficient at 405 nm of 140 m"' cm -' (3). The purification stages and specific activities are shown in Table I. Properties of the Duncated Heme-Heme Oxygenase Complex -The Soret maximum of the femc complex formed when heme is added to purified heme oxygenase and the excess heme is removed by passage through a hydroxylapatite column is at 404 nm (Fig. 2). Reduction of the ferric complex with dithionite under an atmosphere of carbon monoxide yields the ferrouscarbon monoxide complex with a Soret band at 418 nm and well defined a and p bands at 568 and 538 nm, respectively (Fig. 2).
Passage of the carbon monoxide complex through a Sephadex G25 column causes the Soret band to shift to 410 nm, and the (Y and p bands to shift to 574 and 540 nm, respectively, as expected for conversion of the carbon monoxide complex to the ferrous dioxygen complex (Fig. 2). These values are comparable with those reported for the ferrous dioxygen complex of native rat liver heme oxygenase (2).
Catalytic %mover of Duncated Heme Oxygenase-The heme in the ferric heme-heme oxygenase complex is quantitatively converted to biliverdin by addition of NADPH and cytochrome P450 reductase (Fig. 3). Residual heme was not detected by HPLC after the reaction and the product formed had a retention time and absorption spectrum identical to those of authentic biliverdin M a (not shown). The specific activity of the enzyme, determined from the increase in absorbance at 468 nm, was 6000 nmol h-' mg-l, a value identical to that reported for purified rat liver heme oxygenase (2). Turnover of the truncated heme-heme oxygenase complex in the presence of carbon monoxide was examined to determine if the reaction, as in the case of the native enzyme, can be arrested at an intermediate stage by complexation with carbon monoxide. In fact, carbon monoxide causes accumulation of a biliverdin precursor (Amm = 640 nm) spectroscopically identical to that obtained with the native enzyme (Fig. 4u) (21, 23). Furthermore, the spectrum obtained when the intermediate accumulated in the presence of carbon monoxide is treated with pyridine and extracted into chloroform (Fig. 4c) is similar to that reported for similar treatment of the native enzyme (21, 23).
Reaction of Duncated Heme Oxygenase with H20rReaction of the heme-heme oxygenase complex with HzOz was investigated to determine if the peroxide can replace cytochrome P450 reductase, NADPH, and 0, in the formation of biliverdin. Addition of 1 eq of H202 to the heme-heme oxygenase complex resulted in virtual loss of the Soret absorbance and an increase in absorbance at -680 nm (Fig. 4b). At no time did the spectrum suggest formation of a ferryl species. Acidification and extraction with chloroform resulted in loss of the green color and isolation not of biliverdin but of a pink residue tentatively attributed to dipyrrole ("propentdyopent") and monopyrrole porphyrin degradation products (31, 32). However, addition of pyridine to the complex prior to acidification and extraction results in the observation of a normal verdoheme-like spectrum with maxima at 400,504,536, and 680 nm (Fig. 4c) (Fig. 5). The product was identified by direct comparison of its absorption spectrum and HPLC retention time with those of an authentic sample of biliverdin Ma (not shown).
Reaction of Duncated Heme Oxygenase with Alkyl-and Acylperoxides-The ability of alkyl and acylhydroperoxides to replace cytochrome P450 reductase and NADPH in the heme oxygenase reaction was investigated to determine whether the enzyme would tolerate the presence of a substituent on one of the peroxide oxygen atoms. Reaction of the heme-heme oxygenase complex with 1 eq of cumene hydroperoxide, tert-butylhydroperoxide, or mCPBA was found to decrease the intensity of the Soret band without increasing the absorption at 640-680 nm. Reaction with 5 eq of cumene hydroperoxide caused little shift in the Soret maximum, but reaction with 5 eq of mCPBA ) addition of 1 eq of HzOz. The final spectrum is that of the shifted the maximum from 404 to 408 nm (Fig. 6). All the acyland alkylhydroperoxides cause the Soret absorption to diminish with an isosbestic point between 416-420 nm, spectroscopic changes consistent with the formation of a ferryl intermediate. Support for a ferryl intermediate is provided by the observation that addition of phenol or ascorbic acid regenerates the spectrum of the ferric heme-heme oxygenase complex. Furthermore, the formation of a verdoheme-like biliverdin precursor was not observed with the acyl-and alkylhydroperoxides. Thus, the 680-nm verdoheme-pyridine complex observed when pyridine is added after reaction with HzO, is not detected when pyridine is added after reaction with the alkylhydroperoxides. In fact, addition of mCPBA prior to Hz02 prevents the Hz02dependent increase in absorbance at 680 nm associated with normal cleavage of the heme group (not shown).
In addition to the formation of a ferryl species, addition of 1 eq of mCPBA to the ferric heme-heme oxygenase complex produces a weak EPR signal. A strong EPR signal at g = 2.006 is obtained when 5 eq of the peracid are added (Fig. 7). The appearance of the EPR signal parallels the decrease in the Soret band caused by reaction with the peracid. The second oxidation equivalent provided by cleavage of the peroxide thus appears to be dissipated as a protein radical.
Heme Orygenase as Peroxidase-The ability of phenol to regenerate the ferric heme-heme oxygenase complex from the ferryl species produced by reaction with alkylhydroperoxides suggests that the ferryl species catalyzes the oxidation of phenols. In fact, the heme-heme oxygenase complex promotes the peroxidation of guaiacol in the presence of 5 eq of mCPBA (rate = 6.9 nmol mg") or cumene hydroperoxide (rate = 2.2 nmol s-l mg-'), but does not detectably catalyze guaiacol oxidation in the presence of NADPH and cytochrome P450 r e d u c t a~e .~ that remains associated with bacterial membranes and is proteolytically digested to smaller fragments. Similar results were reported during the course of this work by Ishikawa et al. (13). We have overcome these problems by expressing a truncated form of heme oxygenase without the 23 COOH-terminal amino acids that serve as a membrane anchor. A soluble 30-kDa protein was thus obtained that was filly active at a level of approximately 30 mg/liter after purification ( Table I).
Heme binds to the truncated heme oxygenase to give a complex with the same spectroscopic properties as the complex of the native protein in the ferric, ferrous dioxy, and ferrous carbonmonoxy states (Fig. 2). The truncated enzyme, when reconstituted with rat liver cytochrome P450 reductase and NADPH, converts heme to biliverdin (Fig. 3) at the same rate as the native enzyme ( Table I). As also reported for the native enzyme (21,221, incubation of the truncated heme-heme oxygenase complex with cytochrome P450 reductase and NADPH under an atmosphere of carbon monoxide results in the accumulation of an intermediate with an absorption maximum at 638 nm (Fig. 4A). This putative verdoheme-enzyme complex is converted to biliverdin in the presence of excess NADPH if the carbon monoxide is replaced by oxygen. The truncated enzyme is thus catalytically indistinguishable from the native enzyme.
Reaction of the heme-heme oxygenase complex with limited amounts of Hz02 yields an intermediate which, if directly extracted from the complex, decomposes to unidentified heme degradation products. However, if the intermediate is reduced under an atmosphere of carbon monoxide, a species is obtained with the same spectroscopic properties as the verdoheme complex produced by incubation of the heme-heme oxygenase complex with cytochrome P450 reductase and NADPH under an atmosphere of oxygen and carbon monoxide (Fig. 4). Furthermore, if cytochrome P450 reductase and NADPH are added to the enzyme-bound intermediate produced with H202, biliverdin is formed as the principal reaction product (Fig. 5). H20z can thus substitute for catalytically reduced molecular oxygen in meso-hydroxylation of the heme. meso-Hydroxylation, in effect, yields verdoheme because the conversion of a-meso-hydroxyheme to verdoheme requires molecular oxygen but no additional reducing equivalents. In contrast, the conversion of verdoheme to biliverdin clearly requires reduction of the verdoheme-enzyme complex by cytochrome P450 reductase. Hz02 is not a viable substitute for catalytically activated dioxygen in this step of the transformation.
The finding that Hz02 supports heme a-meso-hydroxylation suggests that the hydroxylating species may be a ferryl (for- mally FeV=O) complex generated by heterolysis of the peroxide. This mechanism is suggested by the formation of ferryl complexes in the reactions of HzOz with iron porphyrins (33), peroxidases (341, globins (35,36), catalases (37), and monooxygenases (38). A possible mechanism for transfer of the ferryl oxygen to the a-meso-carbon is a stepwise "walk" of the oxygen from the iron via a pyrrole nitrogen to the meso position (39). To explore this aspect of the catalytic process, we examined the reaction of the heme-heme oxygenase complex with mCPBA, cumene hydroperoxide, and tert-butylhydroperoxide. These peroxides react with hemoproteins to give the same ferryl complex as are obtained with H202 and should therefore support biliverdin formation if a ferryl species is responsible for the reaction (40). However, reaction of the heme-heme oxygenase complex with mCPBA or the alkylhydroperoxides produces a relatively stable species with a spectrum similar to that of the F e W 4 ferryl complex of myoglobin ( Fig. 6) (41). This ferryl complex is one oxidation equivalent above the femc state. A two-electron-oxidized ferryl complex comparable with that of Compound I of horseradish peroxidase (34) is not detected, either because it is not formed or, as suggested by EPR observation of a protein radical (Fig. 7), because it rapidly oxidizes the protein (42). Identification of the complex as a ferryl species is consistent with the finding that it oxidizes guaiacol to colored oligomeric products. In contrast to these results, reaction of the heme-heme oxygenase complex with either H20z or cytochrome P450 reductase and NADPH does not give a detectable ferryl complex, does not catalyze guaiacol peroxidation, and does not cause protein radical formation. The conclusion that a ferryl species is not involved in biliverdin formation is confirmed by the fact that formation of the ferryl complex with mCPBAprevents rather than accelerates the formation of verdoheme when HzOz is subsequently added.
What are the mechanistic alternatives if a ferryl species is excluded? One possibility is that heme hydroxylation involves addition of a hydroxyl radical produced by homolysis of the dioxygen bond in the [Fe-OOH] enzyme complex. A free hydroxyl radical is an unlikely reaction intermediate, however, because the enzyme would have to force this exceedingly reactive species to react exclusively with the meso-carbon rather than with other porphyrin or protein sites. Two alternatives are readily envisioned for reaction of the porphyrin ring with the intact iron-dioxygen complex that lead to cy-meso-hydroxyheme: (a) nucleophilic addition of the terminal oxygen of the unprotonated complex [Fe-00-1 to the a-meso-carbon to give a peroxo-bridged (Fe-O-O"C,,,) intermediate or ( b electrophilic addition of the terminal oxygen of the protonated complex [Fe-OOHI with concomitant cleavage of the dioxygen bond (Scheme 2). The two latter mechanisms rationalize the ability of H202 but not alkylhydroperoxides to replace cytochrome P450 reductase and NADPH in heme hydroxylation, the absence of a ferryl species from the reaction trajectory that inserts the oxygen atom, and the ability of the protein to control the regiospecificity of heme hydroxylation.