Heme Oxygenase-1, Intermediates in Verdoheme Formation and the Requirement for Reduction Equivalents*

(Received for publication, November 20, 1996, and in revised form, January 8, 1997)

Yi Liu Dagger , Pierre Moënne-Loccoz §, Thomas M. Loehr § and Paul R. Ortiz de Montellano Dagger

From the Dagger  Department of Pharmaceutical Chemistry, School of Pharmacy, University of California, San Francisco, California 94143-0446 and the § Department of Chemistry, Biochemistry, and Molecular Biology, Oregon Graduate Institute of Science and Technology, Portland, Oregon 97291-1000

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES


ABSTRACT

Conversion of heme to verdoheme by heme oxygenase-1 (HO-1) is thought to involve alpha -meso-hydroxylation and elimination of the meso-carbon as CO, a reaction supported by both H2O2 and NADPH-cytochrome P450 reductase/O2. Anaerobic reaction of the heme-HO-1 complex with 1 eq of H2O2 produces an enzyme-bound intermediate identified by spectroscopic methods as alpha -meso-hydroxyheme. This is the first direct evidence for HO-1-catalyzed formation of alpha -meso-hydroxyheme. alpha -meso-Hydroxyheme exists as a mixture of Fe(III) phenolate, Fe(III) keto anion, and Fe(II) keto pi  neutral radical resonance structures. EPR shows that complexation with CO enhances the Fe(II) pi  neutral radical component. Reaction of the alpha -meso-hydroxyheme-HO-1 complex with O2 generates Fe(III) verdoheme, which can be reduced in the presence of CO to the Fe(II) verdoheme-CO complex. Thus, conversion of alpha -meso-hydroxyheme to Fe(III) verdoheme, in contrast to a previous report (Matera, K. M., Takahashi, S., Fujii, H., Zhou, H., Ishikawa, K., Yoshimura, T., Rousseau, D. L., Yoshida, T., and Ikeda-Saito, M. (1996) J. Biol. Chem. 271, 6618-6624), does not require a reducing equivalent. An electron is only required to reduce ferric to ferrous verdoheme in the first step of its conversion to biliverdin.


INTRODUCTION

Heme oxygenase catalyzes the NADPH- and cytochrome P450 reductase-dependent oxidation of heme (iron protoporphyrin IX regardless of oxidation and ligation state) to biliverdin and CO (1). This enzyme is of physiological interest because of the biological properties of its reaction products: biliverdin and CO. Biliverdin is normally reduced to bilirubin by biliverdin reductase and is excreted after conjugation with glucuronic acid (2). Excretion of bilirubin is frequently impaired in newborn children and in individuals with a genetic glucuronyltransferase deficiency (3). Unconjugated bilirubin is neurotoxic, and approaches to the prevention of its accumulation, including the inhibition of heme oxygenase, are of potential clinical utility (4-6). More recently, a potentially important but controversial role as a neurotransmitter akin to nitric oxide has been invoked for the CO produced by heme oxygenase (7-10).

Two forms of the enzyme, denoted as HO-1 1 and HO-2, have been identified (11-13). HO-1 is induced by both a variety of chemical agents and a range of stress conditions and is found in the highest concentrations in the spleen and liver. HO-2 is not inducible and is found in the highest concentrations in the brain and testes. HO-1 and HO-2 are membrane-bound proteins with a C-terminal lipophilic domain that anchors the protein to the endoplasmic reticulum (14, 15). We have reported that a 30-kDa truncated version of HO-1 lacking the 23-amino acid C-terminal membrane anchor can be expressed in Escherichia coli in high yields (16, 17). The truncated protein is soluble and retains full catalytic activity. Site-directed mutagenesis studies, in combination with catalytic and spectroscopic analyses, have led to unambiguous identification of His-25 as the proximal heme iron ligand in the HO-1 enzyme-substrate complex (18-23). Recent studies have established that His-132, the conserved residue in the distal side of the heme pocket, plays a role in stabilizing the distal water iron ligand and facilitates, but is not absolutely required for, coupled turnover of the enzyme (24).

Heme oxygenase employs heme as both the prosthetic group and substrate. The oxidation of heme by heme oxygenase is thought to involve sequential alpha -meso-hydroxylation, oxygen-dependent fragmentation of the resulting alpha -meso-hydroxyheme product to verdoheme, and oxidative cleavage of verdoheme to biliverdin (Scheme 1). alpha -meso-Hydroxylation, the first step, requires NADPH-cytochrome P450 reductase-dependent reduction of the iron to the ferrous state, oxygen binding to the reduced iron, and a second one-electron reduction of the oxy-ferrous complex (25). The resulting ferric peroxide (Fe(III)-OOH) complex reacts as an electrophile with the porphyrin ring (16, 26) and incorporates an oxygen atom from molecular O2 into the alpha -meso-hydroxyheme product (27). Although synthetic alpha -meso-hydroxyheme has been shown to be converted to biliverdin IXalpha both by HO-1 (28, 29) and by model systems (30-33), the formation of alpha -meso-hydroxyheme has not been directly demonstrated in the enzymatic reaction, possibly because of its high reactivity with oxygen. Intervention of alpha -meso-hydroxyheme as a true intermediate in the normal catalytic process therefore remains unproven. This shortcoming is important in view of the demonstration that alpha -meso-methyl-substituted heme groups are oxidized to the corresponding alpha -biliverdins without the formation of CO, a reaction that does not involve alpha -meso-hydroxylation of the heme (34, 35).


Scheme 1. Reaction intermediates in the heme oxygenase-catalyzed oxidation of heme to biliverdin. The substituents on the porphyrin are vinyl (V) and propionate (Pr).
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The second step of the catalytic process is elimination of the alpha -meso-carbon and attached hydroxyl as CO (36) concomitant with replacement of the carbon in the ring structure by an oxygen atom (37, 38). However, the nature of the intermediates and the reaction mechanism for this transformation remain unclear. Several studies of the conversion of alpha -meso-hydroxyheme to verdoheme have been carried out with chemical model systems (31, 32, 39-41) as well as systems in which alpha -meso-hydroxyheme has been reconstituted into apomyoglobin (31) or HO-1 (29, 42). These studies indicate that a radical species forms when alpha -meso-hydroxyheme reacts with O2 prior to the formation of verdoheme, but the reduction equivalent requirements for the subsequent reaction are a matter of debate. Matera et al. (42) concluded that 1 reducing eq is required for the HO-1-catalyzed reaction, whereas Sano et al. (31) did not find such a requirement in the myoglobin reaction. Our previous demonstration that NADPH-cytochrome P450 reductase and O2 can be replaced by H2O2 in supporting the HO-1-catalyzed conversion of heme to verdoheme is in accord with the conclusion that exogenous electrons are not required for this reaction (16).

We report here that alpha -meso-hydroxyheme is formed as a stable intermediate when the heme-hHO-1 complex reacts anaerobically with H2O2 and characterize this intermediate by UV-visible, resonance Raman, and EPR spectroscopy. In the presence of CO, alpha -meso-hydroxyheme exists predominantly as a CO-complexed Fe(II) pi  neutral radical. On exposure to O2, this porphyrin radical reacts with O2 to yield Fe(III) verdoheme with no requirement for exogenous reducing equivalents. An electron is only required to reduce Fe(III) to Fe(II) verdoheme.


EXPERIMENTAL PROCEDURES

General Methods

hHO-1 without the 23-amino acid membrane anchor was expressed in E. coli and was purified as previously reported (16, 17). The truncated protein has the same catalytic activity as the full-length form. The catalytic activity was assayed by monitoring the formation of bilirubin at 37 °C in the presence of biliverdin reductase, and the biliverdin products were analyzed by high pressure liquid chromatography as described previously (16).

Chemicals

H2O2 (30%), NADPH, protoporphyrin IX, methyl viologen, sodium dithionite, and pyridine were obtained from either Aldrich or Sigma. High purity argon (99.998%) and CO (99.95%) were obtained from Matheson and Aldrich, respectively. All other chemicals were the highest grade available and were used without further purification.

Anaerobic Reaction of the Heme-hHO-1 Complex with H2O2

A custom-made anaerobic cuvette (glass shop, University of California, Berkeley, CA) was assembled from a fluorescence cuvette with four optical windows (1- and 0.4- or 0.2-cm path length, respectively) by attaching it to a spherical chamber (for gas exchange and mixing) bearing a standard ground-glass joint, a septum-sealed adapter, and a side arm for the addition of reagents after gas exchange is complete. The reconstituted heme-hHO-1 complex (35 µM in 1.2 ml of 100 mM potassium phosphate buffer, pH 7.4) was placed in the UV cuvette, and 1 eq of H2O2 (50 µl) was placed in the side arm. The concentration of H2O2 was quantitated by titration with iodide (43) or by measuring the absorbance at 240 nm (epsilon 240 = 43.6 M-1 cm-1). The protein solution and H2O2 were made anaerobic by flushing the cuvette with oxygen-free argon for at least 40 min. The argon was passed through an oxygen scrubber to remove the trace O2 contamination from the gas, and a water-filled bubbler was used to humidify the argon and to prevent drying of the solutions in the cuvette. During the gas exchange, the protein solution was placed in the round bowl of the cuvette to maximize the surface area and the argon exchange rate. After the solutions were fully anaerobic, the reaction was initiated by mixing the protein solution with the H2O2 in the side arm at 23 °C and recording the UV-visible spectrum until no further changes were observed. For subsequent reaction with O2, the sealed septum was removed; the protein solution was flushed with 100% O2; and the UV-visible spectrum was recorded.

For the experiments performed under an atmosphere of CO, the protein and H2O2 solutions were flushed with O2-free CO. The CO (99.95%) gas was bubbled through a sodium dithionite solution containing reduced methyl viologen (blue) as an indicator to remove oxygen impurities prior to entry into the cuvette. The procedure was otherwise the same as that for the anaerobic experiments.

Resonance Raman Spectroscopy

Resonance Raman experiments were performed using the same anaerobic cuvette used for the absorption spectroscopy. The procedures used to prepare samples for absorption measurements were repeated for resonance Raman experiments with higher protein concentrations (~150 µM). Optical absorption data could be concomitantly obtained using the cuvette with a 0.2-cm path length. Resonance Raman spectra were recorded on a custom McPherson 2061/207 spectrograph (0.67-m focal length, 1800 grooves/mm of grating, and 6 cm-1 spectral resolution) using a Kaiser Optical holographic super-notch filter and a Princeton Instruments liquid N2-cooled CCD detector (LN-1100PB). The excitation source was provided by an Innova 302 krypton laser (413 nm, ~5 mW). Spectra were collected in a 90°-scattering geometry at room temperature with a collecting time of a few minutes. Peak frequencies were calibrated relative to an indene standard and are accurate to ±1 cm-1.

EPR Spectroscopy

Anaerobic sample handling was as described above except that higher concentrations of the reconstituted heme-hHO-1 complex were used for EPR experiments. The protein solution (300 µM, 250 µl) in a Teflon-sealed constantly stirred 1-ml reaction vial was made anaerobic by flushing, as required, with either O2-free argon or CO. The reaction, initiated by anaerobic addition of 1 eq of H2O2, was allowed to proceed for 5 min at 23 °C before the protein solution was transferred to an anaerobic EPR tube, in which it was frozen by immersion in liquid nitrogen. For the reaction with O2, 100% O2 was used to flush the head space over the protein solution with constant stirring. Sodium dithionite was prepared in anaerobic buffered solutions and was quantitated by titration with potassium ferricyanide solution (epsilon 420 = 1.03 mM-1 cm-1). X-band EPR spectra were recorded using a Varian E-109 spectrometer equipped with an Oxford Instruments ESR-910 liquid helium cryostat, a Hewlett-Packard 436A power meter, and a Hewlett-Packard 5350B microwave frequency counter. Temperature, g value calibrations, data acquisition, subtraction, and integration procedures were as described previously (44-46).


RESULTS

UV-visible and Resonance Raman Analyses of the Formation of alpha -meso-Hydroxyheme with H2O2

When the heme-hHO-1 complex (Fig. 1, --) reacts anaerobically (argon atmosphere) with 1 eq of H2O2 at pH 7.4, the Soret band (- · -) becomes broader, decreases in intensity, and crosses at 425 nm that of the original heme-hHO-1 complex. In the visible region, the alpha  and beta  bands at 574 and 536 nm are greatly attenuated; the absorbance at 635 nm increases; and a new band appears around 820 nm. These spectroscopic changes differ dramatically from those observed when verdoheme is formed by aerobic addition of 1 eq of H2O2 to the heme-hHO-1 complex (16). The new species that is formed anaerobically is stable at 23 °C for at least 30 min if anaerobicity is maintained. The broad Soret band at 405 nm and the relatively featureless visible region of the anaerobic intermediate are similar to those of the reconstituted alpha -meso-hydroxyheme-HO-1 complex reported by Matera et al. (42) and the alpha -meso-hydroxyheme-myoglobin complex reported by Sano et al. (31). When the anaerobically generated intermediate is exposed to O2, the Soret intensity undergoes a further slight decrease; the broadening of the Soret band and the band at 820 nm disappear; and the absorption at 660-690 nm increases markedly (Fig. 1, - - -). This spectrum, which is the same as that obtained upon aerobic addition of H2O2 (16), suggests that verdoheme is formed by reaction of the anaerobically generated intermediate with O2. The formation of verdoheme was confirmed by the addition of 20% pyridine to the protein solution, which produced the typical spectrum of the verdoheme-pyridine complex (Fig. 1, - ·· -) (16, 37, 38).


Fig. 1. UV-visible spectroscopic monitoring of alpha -meso-hydroxyheme formation in the anaerobic reaction of the heme-hHO-1 complex with H2O2 and of its conversion to verdoheme by subsequent reaction with O2. In panels I and II are shown the short and long wavelength regions of the spectra of (a) the ferric heme-hHO-1 complex before the reaction (--), (b) the product of the anaerobic reaction of the heme-hHO-1 complex with 1 eq of H2O2 (- · -), and (c) the product obtained from reaction of the sample in spectrum b with O2 (- - -). In panel III is shown the product of spectrum c after the addition of 20% pyridine (- ·· -).
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Additional evidence for the formation of alpha -meso-hydroxyheme in the anaerobic reaction of H2O2 with the heme-hHO-1 complex is provided by comparison of the resonance Raman spectrum of the product with that reported for HO-1 reconstituted with synthetic alpha -meso-hydroxyheme (42). The resonance Raman spectrum of the ferric heme-hHO-1 complex (Fig. 2, trace A), as previously reported (18, 22), is characteristic of a mixture of high and low spin hexacoordinate heme dominated by a nu 4 porphyrin skeletal mode at 1375 cm-1. Anaerobic addition of 1 eq of H2O2 leads to a decrease of the contributions from the ferric heme and the appearance of new resonance Raman features that signal the formation of a new species (Fig. 2, trace B), although the residual band at 1375 cm-1 indicates that some unreacted ferric heme is still present. Reaction with higher concentrations of H2O2 further decreases the ferric heme bands, but no increase is observed in the bands attributed to alpha -meso-hydroxyheme. This is probably due to side reactions of alpha -meso-hydroxyheme with H2O2.2 The difference spectrum (Fig. 2, trace C) obtained by subtracting the spectrum of the residual starting material from trace B exhibits major resonance Raman bands at 889, 1125, 1226, 1334, 1354, 1401, 1581, and 1616 cm-1. Allowing for an experimental error of 2 cm-1, the difference spectrum reproduces the features of the previously reported spectrum of the alpha -meso-hydroxyheme complex (42). Although previous studies have shown that chemically synthesized alpha -meso-hydroxyheme can be converted to biliverdin (29, 31, 42), this is the first demonstration of the heme oxygenase-catalyzed formation of alpha -meso-hydroxyheme.


Fig. 2. Resonance Raman spectra of the alpha -meso-hydroxyheme formed by anaerobic reaction of the heme-hHO-1 complex with H2O2. Trace A, the ferric heme-hHO-1 complex before the reaction; trace B, the same sample after anaerobic reaction with 1 eq of H2O2; trace C, the alpha -meso-hydroxyheme-hHO-1 complex obtained from trace B after subtraction of the spectrum of the unreacted heme-hHO-1 complex. Spectra were recorded at room temperature (413 nm excitation).
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Kinetics of the Formation of alpha -meso-Hydroxyheme

Rapid scanning spectroscopy at 23 °C shows that formation of the alpha -meso-hydroxyheme intermediate in the anaerobic reaction of the heme-hHO-1 complex with H2O2 is complete within 4 min (Fig. 3A). As before, completion of the reaction is associated with a decrease in and broadening of the Soret peak with an isosbestic point at 425 nm and a slight increase in the absorbance at 635 nm. Aerobic reaction of the heme-hHO-1 complex with H2O2 also reaches completion within 4 min at 23 °C (Fig. 3B). However, the isosbestic point at 425 nm and the broadening of the Soret band are not observed, and the 660-690 nm absorption characteristic of verdoheme increases in parallel with the decrease in the Soret band. In the aerobic reaction, the heme appears to be directly transformed into verdoheme without the accumulation of alpha -meso-hydroxyheme. When preformed alpha -meso-hydroxyheme reacts with oxygen, there is a slight loss of Soret absorption and an increase in the 660-690 nm absorption (Fig. 3C). These changes are complete within 5 s at 23 °C, indicating that the conversion of alpha -meso-hydroxyheme to verdoheme is very rapid. The kinetics of these aerobic and anaerobic reactions of the heme-hHO-1 complex with H2O2 indicate that (a) a new stable intermediate with a different UV-visible spectrum forms anaerobically with H2O2; (b) the new intermediate is a precursor of verdoheme and is converted rapidly to verdoheme upon exposure to O2; and (c) the rate of the H2O2-dependent oxidation of heme to verdoheme appears to be limited by the rate of formation of alpha -meso-hydroxyheme. This latter result is consistent with the observation that alpha -meso-hydroxyheme does not accumulate in the aerobic reaction of the heme-hHO-1 complex with H2O2.


Fig. 3. Time course of the anaerobic and aerobic reactions of the heme-hHO-1 complex with H2O2 as monitored by UV-visible spectroscopy. A and B, anaerobic (A) and aerobic (B) H2O2-dependent conversion of the ferric heme-hHO-1 complex to the alpha -meso-hydroxyheme-hHO-1 and verdoheme-hHO-1 complexes, respectively; C, the subsequent aerobic conversion of the alpha -meso-hydroxyheme to the verdoheme-hHO-1 complex. In A and B, a spectrum was recorded every 20 s for 4 min at 23 °C after initiation of the reaction by the addition of H2O2. In C, a spectrum was recorded every 5 s for 30 s at 23 °C.
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Monitoring of the Formation of alpha -meso-Hydroxyheme by EPR Spectroscopy

The resting state of the heme-hHO-1 complex at pH 7.4, as previously reported, gives predominantly a high spin (S = 5/2) ferric axial EPR signal with gperp  = 6 and gpar  = 2 (Fig. 4, trace A) (18, 21). When the complex reacts anaerobically with 1 eq of H2O2, a slight loss of the g = 6 signal and a radical signal at g = 2.008 are observed (Fig. 4, trace B). The deprotonated ferric alpha -meso-hydroxyheme can be represented as a mixture of ferric phenolate, ferric keto anion, and ferrous keto pi  neutral radical resonance forms (32). The EPR data in Fig. 4 (trace B) are consistent with this interpretation: the pi  neutral radical species accounts for the g = 2.008 signal, and the EPR-silent ferrous iron accounts for the decrease in the g = 6 signal. The g = 6 signal is broader (Fig. 4, trace B) than that in the parent ferric state (trace A). The broadening of the signal may be due to either the generation of a rhombic signal contributed by the ferric alpha -meso-hydroxyheme or the presence of a more heterogeneous electronic structure in the keto-enol iron complex. It is difficult to differentiate the signal type due to interference by the g = 6 axial signal from unreacted ferric heme, the presence of which is confirmed by the resonance Raman experiments. When the EPR signal from unreacted ferric heme is subtracted, the remaining spectrum shows a ferric alpha -meso-hydroxyheme rhombic signal (Fig. 4, trace C) consistent with that reported by Matera et al. (42). The rhombic signal suggests that the ferric keto-enol form of alpha -meso-hydroxyheme has a high spin iron (S = 5/2). However, the ferrous pi  neutral radical species at g = 2.008 was not observed previously in either the reconstituted alpha -meso-hydroxyheme-HO-1 (42) or alpha -meso-hydroxyheme-myoglobin (31) system. Strong evidence for the ferrous pi  neutral radical character of alpha -meso-hydroxyheme is provided by the change in the EPR spectrum observed upon complexation of the iron with CO (Fig. 4, trace D). The EPR spectrum of the alpha -meso-hydroxyheme generated with 1 eq of H2O2 under an atmosphere of CO shows that the g = 6 signal is virtually eliminated, whereas the g = 2.008 signal is greatly enhanced (Fig. 4, trace D). The dramatic increase in the g = 2.008 radical species in the presence of CO suggests that CO forms a complex with the ferrous form of the intermediate and thereby stabilizes the Fe(II) keto pi  neutral radical structure relative to the enol and keto anion resonance forms. This inference is consistent with the UV-visible spectrum in Fig. 5, which shows that when alpha -meso-hydroxyheme is formed with 1 eq of H2O2 under an atmosphere of CO, the Soret maximum is red-shifted (from 405 to 408 nm) relative to its position in the absence of CO. The Soret shift argues for the formation of a CO complex of the Fe(II) pi  neutral radical species.


Fig. 4. EPR spectra of the formation of alpha -meso-hydroxyheme in the anaerobic reaction of the heme-hHO-1 complex with H2O2 and the effect of complexation with CO. Trace A, the ferric heme-hHO-1 complex; trace B, the product of the anaerobic reaction of the heme-hHO-1 complex with 1 eq of H2O2; trace C, the alpha -meso-hydroxyheme complex after subtraction of the spectrum of unreacted heme from trace B; trace D, the product obtained anaerobically with 1 eq of H2O2 after complexation with CO. The unreacted heme spectrum (see Fig. 8, trace B) used for subtraction was obtained by exposure of the sample in trace B to O2. The g values are indicated. The instrumental conditions were as follows: scan range, 500 mT; scan center, 250 mT; modulation amplitude, 1 mT; modulation frequency, 100 kHz; microwave frequency, 9.23 GHz; microwave power, 1-µW gain, 1.6 × 104; and temperature, 6 K.
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Fig. 5. Effect of CO on the Soret band of the heme-hHO-1 complex after anaerobic reaction with H2O2. Shown are the Soret band of the ferric heme-hHO-1 complex before the reaction (····) and the Soret band of the heme-hHO-1 complex after anaerobic reaction with 1 eq of H2O2 under an atmosphere of either argon (- ·· -) or CO (--).
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No significant changes are observed in the high frequency region of the resonance Raman spectrum of the alpha -meso-hydroxyheme-hHO-1 complex when CO is added (data not shown). Several explanations are possible for the absence of a change in the resonance Raman spectrum on CO complexation. Although the EPR changes measured at 6 K are dramatic, they may be tempered at high temperature. For example, the shift in the Soret absorption at room temperature is relatively minor. Thus, temperature may affect the population ratio of the different resonance states of the alpha -meso-hydroxyheme-hHO-1 complex. In addition, the formation of radical states on the porphyrin ring is known to greatly decrease their resonance Raman intensity (47). Therefore, the signal for the ferrous pi  neutral radical species may be difficult to detect under the present conditions, where several other species are known to coexist. Moreover, the photolability of heme-bound CO is well known; hence, CO may be dissociated by the laser beam during data collection. The same results were observed when a power of ~1 mW was used, but the light sensitivity of this particular complex is unknown. Similarly, the extent of the resonance Raman changes of the ferrous alpha -meso-hydroxyheme upon CO binding are not known.

Reaction of alpha -meso-Hydroxyheme with O2 Observed by Resonance Raman and EPR Spectroscopy

As already discussed, the 660-690 nm absorption maximum indicative of verdoheme formation increases when O2 is added to the alpha -meso-hydroxyheme generated anaerobically by reaction of the heme-hHO-1 complex with H2O2 (Fig. 1, - - -). Identical experiments were carried out to determine the resonance Raman spectroscopic changes caused by the reaction. The presence of the unreacted ferric heme (nu 4 mode at 1375 cm-1) from the anaerobically generated alpha -meso-hydroxyheme (shown in Fig. 2, trace B) remains unchanged upon exposure to O2. Once the resonance Raman contributions from the unreacted heme have been subtracted (Fig. 6, trace A), the spectrum clearly shows that the bands characteristic of alpha -meso-hydroxyheme (e.g. 889, 1226, 1354, and 1581 cm-1; Fig. 2, trace C) are replaced by a new set of vibrations with intense peaks at 1258, 1463, and 1612 cm-1 (Fig. 6, trace A). We have also recorded the resonance Raman spectrum of the verdoheme-hHO-1 complex generated by aerobic reaction of the heme-hHO-1 complex with H2O2 (Fig. 6, trace B). In this reaction, a higher H2O2 concentration was used, and the conversion of heme to verdoheme is nearly quantitative,3 as indicated by the smaller unreacted heme peak at 1375 cm-1. A perfect match in frequencies is observed between the two spectra (Fig. 6, traces A and B), clearly establishing that verdoheme is formed when the alpha -meso-hydroxyheme complex reacts with O2. An identical resonance Raman spectrum is also obtained when CO-complexed alpha -meso-hydroxyheme reacts with O2, showing that the same verdoheme product is formed in the presence of CO (data not shown). These results demonstrate that oxygen alone is required for the conversion of ferric alpha -meso-hydroxyheme to verdoheme and specifically show that an exogenous electron is not required for this transformation. This finding contradicts the report by Matera et al. (42) that no verdoheme is formed from alpha -meso-hydroxyheme unless an exogenous electron is provided.


Fig. 6. Resonance Raman spectra of the formation of verdoheme by reaction of alpha -meso-hydroxyheme with O2. Trace A, The verdoheme-hHO-1 complex obtained from reaction of the anaerobically generated alpha -meso-hydroxyheme with O2 (sample in Fig. 2 (trace B) after exposure to O2 and subtraction of the resonance Raman spectrum of the unreacted ferric heme); trace B, the verdoheme-hHO-1 complex obtained by aerobic reaction of the ferric heme-hHO-1 complex with 1.5 eq of H2O2; trace C, the reduced verdoheme-hHO-1 complex obtained by anaerobic addition of a stoichiometric amount of dithionite to the sample in trace B. Spectra were recorded at room temperature (413 nm excitation).
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Trace B in Fig. 6 is not identical to that reported by Matera et al. (42) for the Fe(II) verdoheme complex. Major peaks are found at higher frequencies, 1612 (+3) and 1258 (+4) cm-1, and the single broad band at 1463 cm-1 is shown as a pair of bands at 1446 and 1486 cm-1. To determine whether these differences are due to the presence of Fe(III) rather than Fe(II) verdoheme, the resonance Raman spectrum of Fe(II) verdoheme generated by anaerobically adding a stoichiometric amount of dithionite to trace B in Fig. 6 was recorded. A direct comparison of the resonance Raman spectra of the Fe(III) and Fe(II) verdohemes (Fig. 6, traces B and C) reveals several differences. (a) The major bands at 1612 and 1258 cm-1 (Fig. 6, trace B) shift to 1608 and 1252 cm-1, respectively, in the ferrous verdoheme spectrum (trace C); (b) the band at 1463 cm-1 (trace B) splits into two bands at 1441 and 1484 cm-1 in ferrous verdoheme (trace C); and (c) the band at 1353 cm-1 in trace C characteristic of ferrous heme (18) comes from the reduction of the unreacted ferric heme (1375 cm-1 in trace B). This change in the residual heme peak serves to confirm that the iron atoms in the solution are in the ferrous state. Thus, it is seen that the ferrous verdoheme spectrum (Fig. 6, trace C) is essentially identical to that reported by Matera et al. (42). The only significant difference is that the bands at 1338 and 1366 cm-1 in their spectrum are masked in trace C by the unreacted ferrous heme band at 1353 cm-1, but shoulders at higher and lower frequencies are noted. Further support for identification of trace C as that of ferrous verdoheme is provided by the similarities between it and the resonance Raman spectrum of a model Fe(II) verdoheme in pyridine.4 These data prove that the iron in the verdoheme obtained from reaction of alpha -meso-hydroxyheme with oxygen in the absence of reducing equivalents is in the ferric state, as expected.

Another piece of evidence for the formation of a ferric product is provided by the finding that the UV-visible spectrum of the verdoheme complex generated by aerobic reaction of heme-hHO-1 with H2O2 (lambda max = 404, 544, and 680 nm) (Fig. 7, --) remains unchanged when CO is added (····). In contrast, the addition of a stoichiometric amount of dithionite produces ferrous verdoheme (Fig. 7, - ·· -) with a distinct UV-visible spectrum (lambda max = 400, 534, and 690 nm). The addition of CO to the reduced sample yields a UV-visible spectrum with maxima at 412 and 638 nm that clearly identifies the product as the Fe(II) verdoheme-CO complex (Fig. 7, - - -) (16, 37, 38). Thus, reaction of the heme-hHO-1 complex with oxygen in the absence of an external electron source readily yields Fe(III) verdoheme.


Fig. 7. UV-visible spectra of ferric and ferrous verdohemes and of the ferrous CO complex. Shown are the UV-visible spectra of the verdoheme-hHO-1 complex obtained by aerobic reaction of the heme-hHO-1 complex with 1 eq of H2O2 (a) before (--) and (b) after (····) the addition of CO, (c) the ferrous verdoheme-hHO-1 complex obtained by the addition of one electron (via dithionite) to the sample in spectrum a (- ·· -), and (d) the CO-complexed ferrous verdoheme-hHO-1 complex obtained by the addition of CO to the sample in spectrum c (- - -).
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EPR spectroscopy provides additional evidence for the aerobic conversion of alpha -meso-hydroxyheme to Fe(III) verdoheme. When anaerobically generated alpha -meso-hydroxyheme (Fig. 8, trace A; obtained from Fig. 4, trace B) is exposed to O2, the g = 2.008 radical species disappears, and the g = 6 signal decreases (trace B). The remaining g = 6 axial signal comes from the unreacted ferric heme species also observed in the resonance Raman experiments. A fairly weak rhombic signal in the g = 2 region (gz = 2.57, gy = 2.14, and gx = 1.86; gav = 2.19) is also obtained that corresponds to a low spin (S = 1/2) ferric signal. Comparison of this spectrum with that obtained when 1 eq of H2O2 is added aerobically to the heme-hHO-1 complex (Fig. 8, trace C) shows that, in the aerobic reaction, the g = 6 signal is similarly suppressed, and the new low spin ferric iron signal is also generated. However, the residual g = 6 signal due to unreacted heme is smaller (Fig. 8, traces B and C), which suggests that the conversion of heme to verdoheme is more complete when the reaction with H2O2 is carried out aerobically. The loss of the g = 6 axial signal associated with verdoheme formation indicates that Fe(III) verdoheme has a different iron spin state relative to that of ferric heme or alpha -meso-hydroxyheme, possibly a low spin iron (S = 1/2) that gives rise to the rhombic EPR signal at gav = 2.19. The same results, disappearance of the g = 2.008 radical species and appearance of a weak rhombic EPR signal at gav = 2.19, are observed (data not shown) when the CO-complexed ferrous pi  neutral radical (sample in Fig. 4, trace D) is exposed to O2. This finding indicates that the ferrous pi  neutral radical resonance structure of the alpha -meso-hydroxyheme most clearly represents the species that reacts with molecular oxygen. The new gav = 2.19 rhombic EPR signal generated in these reactions suggests that it is a signature of Fe(III) verdoheme, although the reason why it is a weak signal is unclear.


Fig. 8. EPR spectra of the formation of verdoheme by reaction of alpha -meso-hydroxyheme with O2. Trace A, the product of the anaerobic reaction of the heme-hHO-1 complex with 1 eq of H2O2; trace B, the product after the addition of O2 to the sample in trace A; trace C, the verdoheme complex obtained by aerobic addition of 1 eq of H2O2 to the ferric heme-hHO-1 complex. The g = 6 signal corresponds to the unreacted ferric heme-hHO-1 complex. The g values are marked. The instrumental conditions were as follows: scan range, 500 mT; scan center, 250 mT; modulation amplitude, 1 m; modulation frequency, 100 kHz; microwave frequency, 9.23 GHz; microwave power, 1-µW gain, 1.6 × 104; and temperature, 6 K.
[View Larger Version of this Image (17K GIF file)]



DISCUSSION

The first step in heme degradation is thought to be the cytochrome P450 reductase-, NADPH-, and O2-dependent alpha -meso-hydroxylation of heme by heme oxygenase. This transformation requires reduction of the ferric to the ferrous heme-protein complex by an electron from NADPH-cytochrome P450 reductase, oxygen binding to the iron to give the ferrous dioxygen (Fe(II)-OO·) complex, and a second reduction to give an undetected species formally equivalent (after protonation) to a ferric peroxide (Fe(III)-OOH) complex. The ferric peroxide complex then undergoes direct reaction as an electrophile with the pi  bond structure of the heme to give the alpha -meso-hydroxylated heme intermediate (16, 26). The alpha -meso-hydroxyheme is subsequently oxidized to carbon monoxide and enzyme-bound verdoheme. Key evidence for this mechanism is provided by the finding that the conversion of heme to verdoheme is supported by H2O2 in the absence of cytochrome P450 reductase and NADPH (16). Reaction of the heme-hHO-1 complex with H2O2 is arrested at the verdoheme stage even under aerobic conditions because H2O2 is not an appropriate replacement for NADPH-derived electrons and O2 in the conversion of verdoheme to biliverdin.

alpha -meso-Hydroxyhemes have been synthesized, reconstituted into myoglobin and heme oxygenase, and shown to be converted to the corresponding verdohemes and biliverdins (29, 31, 42). alpha -meso-Hydroxyheme, however, has never actually been identified by spectroscopic or other means as an intermediate in the enzymatic conversion of heme to verdoheme. This is a significant shortcoming because the synthetic species could be converted to the observed product without actually being an intermediate in the normal process. Evidence that this is possible is provided by the recent finding that alpha -meso-methyl-substituted hemes are oxidized to give normal alpha -biliverdin, but not CO (34, 35). The kinetic, optical, resonance Raman, and EPR studies of the intermediate formed when 1 eq of H2O2 is added anaerobically to the heme-hHO-1 complex provide the missing evidence that alpha -meso-hydroxyheme is a true intermediate in the catalytic reaction.5

The electronic structure of alpha -meso-hydroxyheme can be presented by three resonance structures: a ferric phenolate ion, a ferric keto anion, and a ferrous keto pi  neutral radical (Scheme 2, 1). The rhombic EPR signal of alpha -meso-hydroxyheme, which represents the contribution of the ferric phenolate and keto resonance structures, suggests that the iron is in the high spin state (S = 5/2). This finding agrees with the results obtained when alpha -hydroxyheme is reconstituted into either HO-1 or apomyoglobin (31, 42). Matera and co-workers (48) and Bogumil et al. (49) also reported that the complex is a five-coordinate species based on the similarity of its rhombic high spin EPR signal to that of pentacoordinate myoglobin mutants. Rhombic high spin signals are often observed for five-coordinate high spin ferric hemoproteins (48, 50). The observation that CO binds to and increases the contribution of (Fig. 4, trace D) the ferrous pi  neutral radical at the expense of the ferric keto-enol resonance forms provides strong support for the electronic ferrous keto-enol neutral radical resonance structure of alpha -meso-hydroxyheme. After exposure of the alpha -meso-hydroxyheme complex to O2, the g = 2.008 radical signal disappears, and verdoheme is formed, in accord with reaction of the radical with O2. The ferrous pi  neutral radical (g = 2.008 signal) was not detected in earlier EPR studies of alpha -meso-hydroxyheme-reconstituted apomyoglobin or HO-1 (31, 42). We have found that the g = 2.008 radical signal is increasingly saturated when the instrument power is increased from 1 to 500 µW at 6 K (data not shown). When the power is raised above 400 µW at 6 K, the radical signal is completely saturated and disappears. It is therefore very likely that, under the conditions (10 K, 1000 µW) used in the earlier studies, the radical signal was saturated and thus was not observed.


Scheme 2. Proposed mechanism for the conversion of alpha -meso-hydroxyheme to verdoheme. Intermediates 7-10 shown in dashed lines represent an alternative pathway for the reaction that is possibly inhibited by CO.
[View Larger Version of this Image (30K GIF file)]


The reaction of alpha -meso-hydroxyheme with O2 to give verdoheme, as shown here by kinetic, optical, EPR, and resonance Raman studies, is rapid and proceeds without a requirement for additional reducing equivalents. This finding is consistent with the reaction mechanism in Scheme 2. The ferrous pi  neutral radical of alpha -meso-hydroxyheme (1) binds O2 on the ring carbon adjacent to the keto group to form a ferrous hydroperoxy radical (O-O·; 2). Intramolecular electron transfer from the iron to the peroxy radical produces a ferric peroxide intermediate (3), the terminal oxygen of which coordinates to the iron to form a peroxo-bridged intermediate (4). Heterolytic dioxygen bond cleavage then yields a ferryl (Fe(IV)=O) species and an alkoxy radical (5) that fragments with the elimination of CO. Internal electron transfer from the resulting pyrrole ring A carbon radical to the ferryl oxygen followed by reaction with the pyrrole ring B carbonyl group produces ferric verdoheme (6). Formation of ferric rather than ferrous verdoheme is established by several lines of evidence. (a) The addition of CO to the verdoheme generated from the heme-hHO-1 complex and 1 eq of H2O2 does not alter the UV-visible spectrum, which suggests that verdoheme is in the ferric state (Fig. 7, ····). (b) The addition of 1 reducing eq and CO to the verdoheme-hHO-1 complex yields the characteristic Fe(II) verdoheme-CO UV-visible spectrum (Fig. 7,- - -). (c) The addition of 1 reducing eq to the verdoheme produces a resonance Raman spectrum (Fig. 6, trace C) that closely resembles that reported for Fe(II) verdoheme (42). (d) The generation of a weak low spin signal associated with loss of the g = 6 signal in the aerobic reaction of the heme-hHO-1 complex with H2O2 suggests that the spin state of the verdoheme intermediate is S = 1/2 Fe(III) (Fig. 8, traces B and C).

Past studies with HO-1 (37, 38, 42) and apomyoglobin (31) suggest that the iron oxidation state of the verdoheme product is Fe(II). For example, Yoshida et al. (37, 38) reported for the NADPH-cytochrome P450 reductase-dependent reaction that a 688 nm intermediate located after hydroxyheme but before iron-biliverdin has a ferrous iron and binds CO with high affinity to yield 638 nm species. However, in all these studies, a source of reducing equivalents, usually NADPH-cytochrome P450 reductase or ascorbate, was present in the reaction. In the presence of reducing equivalents, Fe(III) verdoheme is reduced to Fe(II) verdoheme, which in turn reacts with O2 to give biliverdin unless the high affinity of ferrous verdoheme for CO is used to arrest the reaction. The universal finding that ferrous verdoheme is the product of the reaction of alpha -meso-hydroxyheme with O2 thus simply reflects the fact that exogenous reducing agents were present in the experimental systems that were used. In the present studies carried out without exogenous reducing agents, the reaction product is clearly identified as ferric verdoheme. A case can be made that the formation of Fe(III) verdoheme is desirable to prevent the CO that is released in the reaction from binding to the intermediate and stopping the reaction process.

Sano et al. (31) reported that reaction of the ferric alpha -meso-hydroxyheme-myoglobin complex with O2 yields Fe(III)-biliverdin via Fe(II) verdoheme without the addition of reducing equivalents. Matera et al. (42) reported that both O2 and 1 reducing eq are required for formation of Fe(II) verdoheme from the alpha -meso-hydroxyheme-HO-1 complex. We find that oxygen alone is required for the conversion of alpha -meso-hydroxyheme to Fe(III) verdoheme, although an electron is required to reduce the Fe(III) verdoheme complex to the ferrous state. It is not clear why Matera et al. (42) failed to observe the formation of verdoheme in the reaction of the alpha -meso-hydroxyheme-HO-1 complex with O2. They apparently monitored verdoheme formation under CO by observing the Fe(II) verdoheme-CO complex at lambda max = 402 and 638 nm (37, 38). One possible explanation is that CO binds to the ferrous pi  neutral radical species (see Scheme 2, 7), causing the reaction to stop at the ferrous- CO peroxy radical adduct (8) due to inhibition of electron transfer from the iron to the peroxy radical by the CO ligand. An external electron might then be required to reduce the peroxy radical to the hydroperoxide (9), leading to formation of Fe(II) verdoheme (10) and the conclusion that an exogenous electron is required for the reaction to occur. This scheme would also explain their observation of an organic radical (8) when they exposed the alpha -meso-hydroxyheme-hHO-1 complex to O2 in the presence of CO. However, we have been unable to reproduce their observations by reacting the alpha -meso-hydroxyheme-hHO-1 complex with O2 for 5 min under a 50% CO atmosphere. Although these conditions may not closely reproduce theirs, our observation that the reaction does not stop at the ferrous CO-peroxy radical adduct (8), but continues to Fe(III) verdoheme (6) under these conditions, suggests that the affinity of the peroxy radical species for CO is not high.

Spectroscopically identical verdoheme species are formed when 1 eq of H2O2 is added aerobically to the heme-hHO-1 complex (one step) or by exposure to O2 of the alpha -meso-hydroxyheme complex preformed by anaerobic reaction with 1 eq of H2O2 (two steps). The verdoheme yields obtained by the two approaches differ, however, with a ratio of ~1:0.6 for the one- and two-step processes, respectively. The higher intensity of the g = 6 signal in the EPR spectrum of the two-step reaction suggests that the lower yield of verdoheme is matched by a higher fraction of unreacted heme complex. This finding suggests that the alpha -meso-hydroxyheme intermediate may react with H2O2 to give alternative reaction products, a possibility that is currently under investigation.

In summary, the formation of alpha -meso-hydroxyheme in the HO-1-catalyzed oxidation of heme has been directly demonstrated, and its subsequent conversion to ferric verdoheme has been shown to require O2, but not an exogenous electron. The exogenous electron is only required to reduce ferric verdoheme to the ferrous state.


FOOTNOTES

*   This work was supported by National Institutes of Health Grants DK30297 (to P. R. O. M) and GM34468 (to T. M. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   To whom correspondence should be addressed. Fax: 415-502-4728/476-0688; E-mail: ortiz{at}cgl.ucsf.edu.
1   The abbreviations used are: HO-1, heme oxygenase-1; hHO-1, truncated human HO-1; W, watt(s); T, tesla.
2   When >1 eq of H2O2 is used, anaerobic reaction of the ferric heme-hHO-1 complex with H2O2 yields products in addition to alpha -meso-hydroxyheme. The anaerobically generated alpha -meso-hydroxyheme possibly reacts with H2O2 to give secondary products.
3   In contrast to what is observed when the reaction is carried out anaerobically, in the aerobic reaction of the ferric heme-HO-1 complex with H2O2, complete conversion to verdoheme can be achieved by increasing the H2O2 concentration (1.5 eq of H2O2 was used in this particular experiment).
4   J. Sun, T. M. Loehr, and C. K. Chang, unpublished results.
5   Efforts to detect the alpha -meso-hydroxyheme intermediate using NADPH-cytochrome P450 reductase and stoichiometric amounts of O2 have failed, presumably because oxygen reacts faster with alpha -meso-hydroxyheme than it does with heme. Attempts to add 1 reducing eq "anaerobically" to the preformed oxy-ferrous heme complex yielded a product with UV-visible and EPR spectra similar to those of alpha -meso-hydroxyheme (data not shown), but the reaction product was not homogeneous.

Acknowledgment

We thank Professor John Lipscomb (University of Minnesota) for providing us with access to the EPR spectrometer.


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