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J. Biol. Chem., Vol. 276, Issue 33, 30885-30891, August 17, 2001

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Stimulation of Cytochrome P450 Reactions by Apo-cytochrome b₅

EVIDENCE AGAINST TRANSFER OF HEME FROM CYTOCHROME P450 3A4 TO APO-CYTOCHROME b₅ OR HEME OXYGENASE^*

Hiroshi Yamazaki, Tsutomu Shimada^§, Martha V. Martin^¶, and F. Peter Guengerich^¶

From the  Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa 920-0934, Japan, the ^§ Osaka Prefectural Institute of Public Health, Osaka 537-0025, Japan, and the ^¶ Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

Received for publication, May 31, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Many cytochrome P450 (P450)-dependent reactions have been shown to be stimulated by another microsomal protein, cytochrome b₅ (b₅). Two major explanations are (i) direct electron transfer from b₅ and (ii) a conformational effect in the absence of electron transfer. Some P450s (e.g. 3A4, 2C9, 17A, and 4A7) are stimulated by either b₅ or b₅ devoid of heme (apo-b₅), indicating a lack of electron transfer, whereas other P450s (e.g. 2E1) are stimulated by b₅ but not by apo-b₅. Recently, a proposal has been made by Guryev et al. (Biochemistry 40, 5018-5031, 2001) that the stimulation by apo-b₅ can be explained only by transfer of heme from P450 preparations to apo-b₅, enabling electron transfer. We have repeated earlier findings of stimulation of catalytic activity of testosterone 6-hydroxylation activities with four P450 preparations, in which nearly all of the heme was accounted for as P450. Spectral analysis of mixtures indicated that only ~5% of the heme can be transferred to apo-b₅, which cannot account for the observed stimulation. The presence of the heme scavenger apomyoglobin did not inhibit the stimulation of P450 3A4-dependent testosterone or nifedipine oxidation activity. Further evidence against the presence of loosely bound P450 3A4 heme was provided in experiments with apo-heme oxygenase, in which only 3% of the P450 heme was converted to biliverdin. Finally, b₅ supported NADH-b₅ reductase/P450 3A4-dependent testosterone 6-hydroxylation, but apo-b₅ did not. Thus, apo-b₅ can stimulate P450 3A4 reactions as well as b₅ in the absence of electron transfer, and heme transfer from P450 3A4 to apo-b₅ cannot be used to explain the catalytic stimulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

P450¹ enzymes are widespread in nature and well known for their catalytic versatility (2-5). Interest in these mammalian enzymes is strong because of their critical roles in the metabolism of drugs, steroids, and carcinogens (2, 3, 5, 6). The most common mode of catalysis is mixed-function oxidation, which involves the use of both electrons and O₂ as substrates.

Electron input from pyridine nucleotides into P450s usually follows one of two general modes. The first, which is common in some bacterial and mitochondrial P450s, involves transfer of electrons from a flavoprotein to an iron-sulfur protein to P450 heme. The other major reaction, which is common in the microsomal P450s, involves electron transfer from a 2-flavin flavoprotein (NADPH-P450 reductase) into the P450 heme. Some P450-catalyzed reactions are stimulated by b₅, as first discovered in a series of studies in which NADH stimulated NADPH-dependent reactions in liver microsomes (7). The role of b₅ in some reactions was later reinforced in reconstitution (8-10) and immunochemical inhibition experiments (11). The occurrence and mechanism of b₅ involvement in P450-catalyzed reactions have been studied extensively in the past three decades (12-17). Some P450 reactions involve b₅, but others do not, even with the same P450 enzyme (10, 18). An added complexity is that NADPH-P450 reductase reduces b₅ efficiently as well as P450s, rendering the path of electron transfer difficult to establish in most cases. In general, direct electron transfer to ferric P450 is inefficient because of the unfavorable difference in oxidation potentials (19, 20). Until recently, the generally accepted mechanism for b₅ stimulation has been introduction of an electron into the P450 FeO complex, which is considered to have a much higher oxidation-reduction potential than the P450 Fe³⁺/Fe²⁺ couple (19-22). Early studies with liver microsomes showed changes in the redox state of b₅ (7, 23). Other early experiments with b₅ substituted with redox-inactive metals were also used to argue in favor of an electron transfer mechanism. However, this issue has been difficult to address directly because of the kinetic complexities in mixing b₅ with P450 in the P450 FeO form (24-26).

In 1995, we reported that the reduction of ferric P450 3A4 required the presence of b₅ for maximum rates, as well as a substrate (testosterone) (27). The following year, in the course of doing what were intended to be controls for other experiments, we found that apo-b₅ was as effective as b₅ in stimulating P450 3A4-catalyzed testosterone 6-hydroxylation and nifedipine oxidation (22). We extended this work and reported that P450 2C9 reactions were stimulated by apo-b₅ as well as b₅, but with P450 2E1, only b₅ was stimulatory (28). Other laboratories reported the stimulation of P450s 17A (29) and 4A7 (30, 31) by apo-b₅. Recent work in our own groups has also shown stimulation of catalytic activities of P450s 2A6, 2B6, 2C8, 2C19, and 3A5 by both b₅ and apo-b₅.² (Some P450s have not shown effects of b₅ or apo-b₅, i.e. 1A1, 1A2, 1B1, and 2D6).²

The conclusion regarding these experiments with apo-b₅ is that electron transfer (to P450) by b₅ is not involved in the stimulation (22, 29, 31). The effect of b₅ varies depending upon the particular P450 3A4 system. Enhancement of some (but not all (27, 32)) catalytic activities is seen with either recombinant P450 3A4 or the enzyme purified from liver microsomes (33, 34). The extent of stimulation does vary in different systems, and the reconstituted systems with higher activities appear to show less stimulation (22, 35). However, the effect is usually 2-4-fold. Bacterial or baculovirus-based insect cell membranes in which P450 3A4 and NADPH-P450 reductase are simultaneously expressed have high catalytic activities (including testosterone 6-hydroxylation activity) in the absence of b₅ (25, 36), although b₅ can be added to the membranes to further stimulate activity (37). The critical issue here is what the role of b₅ is in liver (or other tissues), and studies with anti-b₅ antibodies have shown a strong contribution of b₅ to catalytic activity in liver microsomes, regardless of the mechanism (33, 35).

The conclusions reached with apo-b₅ may or may not be relevant to microsomes. However, these stimulatory effects have been observed with numerous purified P450 preparations (3A4, 2C9, 17A, and 4A7) (22, 28-31), and conclusions about modulation of catalytic activities are not surprising in light of our current general understanding of protein-protein interactions. Some P450s have even shown catalytic stimulation by other P450s (28, 38). However, very recently, Guryev et al. (39) have questioned the conclusions of the apo-b₅ studies (22, 29-31, 37). They repeated the experiments cited above with P450s 3A4 and 17A and also found stimulation of catalytic activities by apo-b₅. Some transfer of heme to apo-b₅ was also found; the issue of whether the source was the P450 itself (3A4 or 17A) or adventitiously bound heme was unresolved. These investigators also added heme to purified apo-b₅ and apomyoglobin and characterized the binding as rapid and tight. A key experiment was the subsequent use of apomyoglobin as a heme scavenger in blocking stimulation by apo-b₅ (39).

The results of Guryev et al. (39) are not in accord with the previously held general view about the lack of electron transfer in the stimulation of some reactions by apo-b₅. Concerns involve the report of the presence of excess heme (30%?) in the preparations and the relatively low fraction of transfer of heme (~ 10%) (39). We address some of the issues raised in that work, those that bear directly on the issues of how much heme transfer contributes to the stimulation of P450 activities by apo-b₅ and b₅. Other experiments presented (39) do not directly address the question and are not considered here (e.g. binding to P450, truncations, mutants and use of alternate metals, and reduction in the absence of substrate); differences between apo-b₅, b₅, and b₅ derivatives may seem subtle but are known to have cooperative structural effects (40) that may not be readily interpreted.

We considered a set of P450 3A4 preparations made since the earlier work (22, 28) because our purification methodology has been changed. In these preparations we account for nearly all of the measured heme as P450, although some key experiments were done with a preparation of one of the stocks in which 30% of the P450 had converted to cytochrome P420. Our earlier reported stimulatory effects of apo-b₅ were fully repeatable, and we proceeded to focus on events that could occur in the time frame used to see the stimulations, e.g. reconstitution and assay. The transfer of 3-5% of the heme from P450 3A4 preparations does occur with apo-b₅ or the enzyme heme oxygenase, with conversion of heme to biliverdin in the latter case. This fraction is attributed to trace cytochrome P420 and adds evidence to the view that heme oxygenase, which is normally involved in the physiological degradation of free heme, does not act directly on P450 heme. The lack of an effect of highly purified apomyoglobin as a heme scavenger and the inability of apo-b₅ to support NADH-b₅ reductase/P450 3A4-catalyzed testosterone 6-hydroxylation provide more evidence against an obligatory role for heme transfer to apo-b₅ in the stimulations of P450s.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Chemicals-- Biliverdin (IX) was purchased from ICN (Costa Mesa, CA). Horse apomyoglobin (sequencing grade) was obtained from Sigma Chemical Co.

Spectroscopy and HPLC-- UV-visible spectra were recorded using OLIS-Cary 14 and -Amino DW2 instruments (On-Line Instrument Systems, Bogart, GA). HPLC was done with a Spectra-Physics 8700 pumping system and ThermoSeparation 6000 rapid scanning monochromator or a JASCO PU-980 pumping system (Japan Spectroscopic Co., Tokyo, Japan) and Chromatopac C-R3A detector (Shimadzu, Kyoto, Japan).

Enzymes-- The original P450 3A4 Escherichia coli pCW expression vector (41, 42) was modified to include a C-terminal (His)₅ tag (43). Purification was done using DEAE and Ni²⁺ affinity chromatography as described by Hosea et al. (44). Rat NADPH-P450 reductase was expressed in E. coli (45) and purified as described elsewhere (46, 47). Rabbit liver b₅ was prepared as described previously (48, 49) and used to prepare apo-b₅ (22), which had spectral properties similar to those reported previously (22). Rabbit liver NADH-b₅ reductase was prepared as described previously (50) and had a specific activity of 24.3 µmol K₃Fe₃(CN)₆ reduced min¹ (nmol reductase)¹ (gift of Y. Imai; University Osaka Prefecture, Osaka, Japan).

E. coli-expressed human heme oxygenase (devoid of 23 residues at the C terminus) (51) was a gift of K. Auclair and P. R. Ortiz de Montellano (University of California, San Francisco, CA). The heme in the sample (36 nmol of enzyme) was converted to biliverdin by incubation with an equal concentration of NADPH-P450 reductase in the presence of 0.10 M potassium phosphate buffer (pH 7.4) and an NADPH-generating system (vide infra). Spectral analysis indicated that the reaction was complete in the first minute. The mixture (in 3 ml) was applied to a 1.2 × 60-cm Sephadex G-10 column equilibrated with 50 mM potassium phosphate (pH 7.4) containing 0.10 mM EDTA and eluted with the same. The void volume fraction was detected by A₂₈₀ measurements; spectral analysis indicated the apparent presence of stable NADPH-P450 reductase flavin semiquinone. The sample was concentrated (to 1.9 ml) using an Amicon-Millipore ultrafiltration device equipped with a PM-10 membrane (Millipore Corp., Bedford, MA). Protein analysis indicated nearly complete recovery of the apo-heme oxygenase and NADPH-P450 reductase. This preparation of apo-heme oxygenase was used with hemin chloride, and a preliminary spectral analysis indicated that it was capable of forming biliverdin. NADPH-P450 reductase was added to the apo-heme oxygenase preparation in subsequent work with P450 3A4.

Reconstitution of P450 3A4 and Assay of Testosterone 6-Hydroxylation-- The following order of mixing was used (all concentrations are for a final incubation mixture in 0.25 ml): P450 3A4 (20 nM), NADPH-P450 reductase (40 nM), b₅ or apo-b₅ (0-40 nM, usually 20 nM unless stated otherwise), apomyoglobin (10-40 nM, when indicated), sodium cholate (0.5 mM), and a phospholipid mixture (20 µg/ml) consisting of a 1:1:1 (w/w/w) mixture of L--dilauroyl-sn-glycero-3-phosphocholine, L--dioleyl-sn-glycero-3-phosphocholine, and bovine brain phosphatidylserine (34); these components were mixed in concentrated solution before dilution and allowed to stand for 10 min at room temperature, followed by the addition of potassium phosphate buffer (to 100 mM; pH 7.4), MgCl₂ (to 5 mM), testosterone (to 200 µM), and an NADPH-generating system consisting of 5 mM glucose 6-phosphate, 0.5 mM NADP⁺, and 0.5 unit of glucose 6-phosphate dehydrogenase ml¹. Incubations proceeded for 10 min at 37 °C. Reactions were terminated, and the products were analyzed by HPLC (octadecylsilane column; A₂₄₀; 1.0 ml min¹ flow rate; 64% CH₃OH in H₂O (v/v)).

Analysis of Heme and Biliverdin-- Heme was estimated using the pyridine hemochrome method, with _557-575 = 32.4 mM¹ cm¹ (52, 53). Biliverdin was extracted from incubations with acetone-HCl and analyzed by HPLC as described by Bonkowsky et al. (54), using a Zorbax octadecylsilane column (3 µm; 6.2 × 80 mm; MacModd, Chadds Ford, PA) and both 670 and 405 nm measurements.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS AND DISCUSSION REFERENCES

Stimulation of P450 3A4 Catalytic Activity by Apo-b₅-- Since our first report of the stimulation of P450 3A4 activities by b₅ and apo-b₅ (22), the preparation of recombinant P450 3A4 has been changed in this laboratory. A C-terminal (His)₅ tag had been incorporated to facilitate purification (44). Purification of P450s is done in the absence of nonionic detergents because these compounds have been found to be substrates for P450s, particularly P450 3A4 (55). Four preparations were used in this work. All had been prepared during the time period 1998-2001 and stored at 20 °C. All were relatively low in P420, as indicated in some of the figures presented later (P420 indicates spectrally distinct denatured P450 or loosely bound heme). Analysis of these preparations for P450 (_450-490 = 91 mM¹ cm¹ for Fe²⁺·CO versus Fe²⁺) and heme (pyridine hemochrome _557-575 = 32.4 mM¹ cm¹) gave the following respective concentrations in the preparations: 21.3 µM P450 and 21.6 µM heme (Fig. 1A); 16.0 µM P450 and 14.1 µM heme (Fig. 1B); 34.4 µM P450 and 34.6 µM heme (Fig. 1C); 69.3 µM P450 and 67.7 µM heme (Fig. 1D). Thus, the heme appears to be present nearly completely as P450, assuming the accuracy of these extinction coefficients (52, 53) and allowing for what is probably ~5% error in replicate measurements.

View larger version (26K): [in this window] [in a new window]   Fig. 1.   Stimulation of testosterone 6-hydroxylation activity of four different P450 3A4 preparations by b5 and apo-b5. b5, ; apo-b5, . See "Results and Discussion" for the concentrations of P450 and heme in each preparation.

With all four of these P450 3A4 preparations, b₅ and apo-b₅ both stimulated testosterone 6-hydroxylation nearly equally (Fig. 1). The extent of stimulation was 3-4-fold, similar to that reported earlier with other preparations of these proteins (22) and in the report of Guryev et al. (39).

Maximum stimulation of testosterone 6-hydroxylation was achieved with a ratio of ~1:1 b₅ or apo-b₅ to P450, similar to that seen earlier with P450 3A4-catalyzed testosterone 6-hydroxylation (22) and some catalytic activities of P450 2C9 (28). For unknown reasons, the stimulation of P450 3A4-catalyzed nifedipine (ring) oxidation is maximally stimulated by lower ratios of b₅ or apo-b₅ to P450 (22, 28). It is conceivable that only a fraction of the P450 3A4 binds nifedipine and participates in the reaction (56), but we have no direct evidence to support this possibility.

Analysis of Movement of Heme into b5-- The proposal of Guryev et al. (39) regarding the transfer of heme from P450 3A4 or heme adventitiously bound to P450 3A4 into apo-b₅ was considered because that work showed that the incorporation of heme into apo-b₅ was a rapid process. However, consideration of the known three-dimensional structures of prokaryotic P450s and P450 2C5 indicates that the heme prosthetic group is embedded inside of the protein (57, 58). Rapid loss of heme from P450s would appear to be highly unlikely, given the known thiolate axial ligation. A rapid exchange of P450 3A4 heme with its environment would make the purification of the holoenzyme nearly impossible.

If heme transfer to apo-b₅ is the explanation for the stimulation of P450 3A4 reactions by apo-b₅ (39), then the transfer must be rapid because the reconstitution/assay process is complete within 10-20 min. We restricted most analyses to this time frame (<30 min). The spectra presented in Fig. 2 were recorded after incubations of all system components (except NADPH) under the normal reconstitution conditions. The spectra obtained with apo-b₅ (Fig. 2, C and D) match those obtained in the absence of b₅ (Fig. 2, A and B) and not those obtained with b₅ (Fig. 2, E and F), as judged by the absence of the peaks at 424 and 550 nm. It should also be noted that the P450 spectrum was not decreased in the presence of apo-b₅ (Fig. 2D).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Lack of formation of b5 from apo-b5 in reconstituted testosterone hydroxylation systems. The incubation mixture (1.0 ml) included P450 3A4 (2.0 µM) NADPH-P450 reductase (3.0 µM), a 1:1:1 mixture (w/w/w) of L--1,2-dilauroyl-sn-glycero-3-phosphocholine, L--1,2-dioleoyl-sn-glycero-3-phosphocholine, and bovine brain phosphatidylserine (40 µg ml1), sodium cholate (1.0 mM), potassium HEPES (50 mM, pH 7.4), MgCl2 (30 mM), and testosterone (200 µM). This incubation was used in A and B. In C and D, apo-b5 (2.0 µM) was included; in E and F, b5 (2.0 µM) was included. After mixtures stood at room temperature for at least 15 min, each sample was diluted with 1.0 ml of 0.10 M potassium phosphate buffer containing 1.0 mM EDTA, 40% glycerol (v/v), 0.5% sodium cholate (w/v), and 0.4% Emulgen 911 and divided between two cuvettes. In A, C, and E, a baseline was recorded, and then solid Na2S2O4 was added to the sample cuvette; spectra were recorded (Fe2+ versus Fe3+). In B, D, and F, CO gas was bubbled through the sample cuvette for 60 s, and a baseline was recorded. Na2S2O4 was added to the sample cuvette, and spectra were recorded (Fe2+·CO versus Fe3+). The spectra did not change significantly for C and D after preincubation for 15-75 min before obtaining spectra.

However, the transfer of traces of heme into b₅ might not have been detected in the design used in Fig. 2. We also used a protocol employed by Guryev et al. (39) in which a spectrum of an S₂O-reduced mixture of P450 3A4/b₅ was recorded versus S₂O-reduced P450 3A4 (Fig. 3). In this assay, a small peak at 424 nm was detected when a mixture of apo-b₅ and P450 3A4 was reduced, possibly indicating b₅ formation. The absorbance of this peak is ~5% of that seen when a b₅ solution was used at the same concentration.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Trace formation of b5 from apo-b5 in the presence of P450 3A4. A baseline was recorded between two identical samples of P450 3A4 (2.0 µM) in the same buffer-ligand-cholate mixture described in the Fig. 2 legend. Either b5 (a; 2.0 µM) or apo-b5 (b; 2.0 µM) was added to the reference cuvette. Solid Na2S2O4 was added to both cuvettes, and spectra were recorded.

We conclude that a finite but small amount of b₅ might have been formed due to transfer of heme to apo-b₅. In considering the results presented in Fig. 1, 5% incorporation of heme into apo-b₅ cannot explain the stimulatory effects of apo-b₅ because the amounts of b₅ and apo-b₅ needed to achieve a given amount of stimulation are nearly identical.

Lack of Function of Apo-b5 in NADH-b₅ Reductase/P450 3A4-catalyzed Testosterone 6-Hydroxylation-- Some P450 reactions can be supported with NADH-b₅ reductase and b₅ substituted for NADPH-P450 reductase, with slower rates (59). In these systems, b₅ is required because NADH-b₅ reductase cannot directly transfer electrons to P450s, including P450 3A4 (35). We previously showed that P450 3A4 can catalyze b₅-dependent testosterone 6-hydroxylation in such a system (35) but that apo-b₅ did not substitute for b₅ (22).

This experiment was repeated with new proteins, and the results were identical (Fig. 4). If heme were transferred from P450 3A4 or anything in the P450 3A4 preparation to convert apo-b₅ to b₅, the apo-b₅ should have been functional in this reaction. This is clearly not the case here, with the limit of ~5% contribution (Fig. 4).

View larger version (13K): [in this window] [in a new window]   Fig. 4.   Lack of support of NADH-b5 reductase-dependent P450 3A4-catalyzed testosterone 6-hydroxylation by apo-b5. The standard testosterone hydroxylation system was used (0.20 µM P450 3A4), except that NADPH-P450 reductase and the NADPH-generating system were replaced by NADH-b5 reductase (0.40 µM) and 1.0 mM NADH. A, no b5 added; B, apo-b5 (0.20 µM) added; C, b5 (0.20 µM) added. T, testosterone, T6OH, 6-hydroxy-testosterone (tR 3.45 min, distinct from the tR 3.33 min peak observed in A and B but obscured by T6OH in C). The reaction measured in C corresponds to a rate of 0.67 nmol product formed min1 (nmol P450 3A4)1.

Lack of Inhibition of P450 3A4-supported Reactions by Apomyoglobin-- One of the critical arguments used by Guryev et al. (39) to support the view of the necessary transfer of heme from P450 3A4 to apo-b₅ was the inhibition of apo-b₅ stimulation of P450 3A4-catalyzed testosterone 6-hydroxylation by apomyoglobin. Apomyoglobin rapidly binds heme in solution and was used as a scavenger. Guryev et al. (39) reported that apomyoglobin, added at the concentration of b₅ or apo-b₅, inhibited the stimulation of catalytic activity by apo-b₅ but not by b₅.

We examined this phenomenon with both testosterone 6-hydroxylation and nifedipine oxidation because of the possible significance of the report (Fig. 5). The two P450 3A4 preparations used were those from Fig. 1, but in Fig. 5, B and D, we used a stock in which the P420 content was 30% of the total P450 plus P420 to investigate the possibility that potentially looser binding of heme might facilitate the heme transfer process proposed by Guryev et al. (39).

View larger version (30K): [in this window] [in a new window]   Fig. 5.   Lack of inhibition of P450 3A4-catalyzed oxidations by apomyoglobin with four different P450 3A4 preparations. The systems contained either no b5 or apo-b5 (), b5 (), or apo-b5 (). A and B, testosterone 6-hydroxylation; C and D, nifedipine oxidation. The same P450 3A4 preparation was used in A and C, and a different preparation was used in B and D.

The apomyoglobin:P450:apo-b₅ or b₅ ratios were varied from 0:1:1 to 2:1:1 (Fig. 5). No significant inhibition of basal, b₅-stimulated, or apo-b₅-stimulated testosterone 6-hydroxylation or nifedipine oxidation was observed.

The clear difference with the result reported by Guryev et al. (39) is unexplained. Clearly one of the most significant differences in the systems studied here and in that work is the matter of the extra heme in the preparation reported by those authors (39). As indicated above, one of the stocks was picked to be one in which P420 was increased (probably due to use with repeated thawing and refreezing), but similar results were obtained (Fig. 5, B and D). Sigma sequencing grade horse apomyoglobin was utilized in our work (this is a standard for optimization of amino acid sequenators). Our work was all done with rabbit b₅ and apo-b₅ instead of human apo-b₅ (39), although the stimulation of P450 activities by b₅/apo-b₅ has been demonstrated with proteins from varying sources (22, 29, 31), and the difference is not expected to account for the varying results.

Interaction of Heme Oxygenase with Heme Bound to P450 3A4-- In the early literature involving the enzyme heme oxygenase (the enzyme that degrades free heme to biliverdin IX and CO), the inference was often made that heme oxygenase degrades the heme in P450 (60, 61). Metals such as Co²⁺ induce heme oxygenase and decrease total P450 in rats, but the relationship of these events is not causal. We reported that partially purified hog spleen heme oxygenase has little if any catalytic activity toward purified (rat) P450 2B1 (62). Kutty et al. (63) reported that P450s 2B1 and 1A1 are substrates for rat heme oxygenase, with heme being converted to biliverdin. However, the P450 preparations contained P420, and the assays were done in the presence of detergents (Emulgen 911) in the absence of glycerol, conditions long known to convert P450 to P420 (64). Under these conditions, 35% of the added P450 2B1 was destroyed, and most (70%) was reported to be converted to biliverdin. However, the obligate conversion of P450 to P420 in this process could not be ruled out, as admitted by the authors (63). In the context of current knowledge of the crystal structures of P450 and the position of the heme, the direct access of P450-bound heme to heme oxygenase appears to be highly unlikely. As discussed earlier, the facile exchange of P450 heme with its medium also appears to be highly unlikely.

The analysis of heme loosely bound to heme proteins is not trivial, including P450s. Kutty et al. (63) reported that the Fe²⁺·CO versus Fe²⁺ difference spectra of free heme, heme albumin (bovine), heme added to P450 2B1, and P420 had discernable spectra with wavelength maxima at 409, 414, 415, and 420 nm, respectively. However, we detected wavelength maxima for free heme, methemalbumin, and heme added to P450 3A4 at 413, 413, and 422 nm, respectively (using the peak finder or derivative function of the OLIS software). As discussed some time ago (64), release of heme from its thiol ligation results in the loss of a P450 spectrum (450 nm for Fe²⁺·CO) and the appearance of a band in the region of ~420 nm. The term P420 is rather operational, and spectra do not discern among P420 proteins with heme bound at various sites, inside and on the outside of proteins (see also Ref. 65 regarding the variablity of spectra of P420 reconstituted from apo-P450 2B4 and heme).

If P450 3A4 were able to freely exchange heme with the medium or had excess heme that could be accessible to apo-b₅ or apomyoglobin, then this should also be accessible to heme oxygenase, which would produce biliverdin in the presence of NADPH-P450 reductase and NADPH. We therefore decided to utilize heme oxygenase as a heme scavenger, similar to apomyoglobin. However, interpretation of the catalytic inhibition with high concentrations of heme oxygenase would be complicated by the competition of heme oxygenase with P450 for NADPH-P450 reductase, and we restricted our analysis to the destruction of P450 heme and formation of biliverdin. To increase the sensitivity of the assays, we converted the heme bound to heme oxygenase into biliverdin in the presence of NADPH-P450 reductase and prepared apo-heme oxygenase using gel filtration and ultrafiltration, yielding a preparation that had activity toward free heme.

Incubation of apo-heme oxygenase, NADPH-P450 reductase, and NADPH with P450 3A4 yielded little if any change in the P450 spectrum (Fig. 6). These results argue that the heme in P450 3A4 is not readily accessible to heme oxygenase.

View larger version (24K): [in this window] [in a new window]   Fig. 6.   Lack of effect of incubation of P450 3A4 with apo-heme oxygenase on spectra. P450 3A4 (10.8 µM) was incubated (volume = 260 µl) with apo-heme oxygenase (nominal 7.3 µM), NADPH-P450 reductase (11.5 µM), potassium phosphate buffer (100 mM, pH 7.4), E. coli superoxide dismutase (2 µM), bovine erythrocyte catalase (800 IU ml1), and the NADPH-generating system for 20 min at 23 °C (a). The reaction was diluted with 1.74 ml of 0.10 M potassium phosphate buffer containing 1.0 mM EDTA, 40% glycerol (w/v), 0.5% sodium cholate (w/v), and 0.4% Emulgen 911 (w/v), and the mixture was divided into two cuvettes. CO was bubbled through the sample cuvette for 60 s, and solid Na2S2O4 was added to both cuvettes. The procedure was repeated as above without apo-heme oxygenase (b) or without NADPH (c).

The difficulty in discerning the nature of heme bound in P420 preparations has been discussed above. We incubated P450 3A4 with apo-heme oxygenase, NADPH-P450 reductase, and NADPH for 30 min at 37 °C (similar to Kutty et al. (63), but in the absence of detergent) and analyzed extracts for the product biliverdin. The method (54) was very sensitive, and a few picomoles of biliverdin could be detected. A peak with the expected t_R and the expected ratio of A₆₇₀:A₄₀₅ was detected (Fig. 7). This peak was not seen in control experiments devoid of NADPH or apo-heme oxygenase and accounts for 3% of the heme in the P450 preparation (mean of duplicate experiments). This loss would, of course, not have been detectable in Fig. 6.

View larger version (22K): [in this window] [in a new window]   Fig. 7.   Formation of trace biliverdin from a P450 3A4 preparation in the presence of apo-heme oxygenase. Mixtures were prepared as described in the Fig. 6 legend and incubated for 30 min at 37 °C. A, HPLC of 140 pmol of standard biliverdin IX; B, HPLC of 40% (200 µl) of a sample of the complete system containing P450 3A4 (3.5 nmol of P450), apo-heme oxygenase, NADPH-P450 reductase, and the NADPH-generating system; C, HPLC of 40% of an incubation similar to that in B but devoid of P450 3A4. The traces are at 405 and 670 nm (the same tR values are offset 0.5 min in presentation due to the HPLC software).

We conclude that, as expected, P450 3A4 is a poor substrate for heme oxygenase, and this is probably the case for all intact P450s. We also conclude that the small amount of biliverdin formed in the presence of a high concentration of heme oxygenase is further evidence that only trace amounts of loosely bound heme are associated with these P450 3A4 preparations. The biliverdin that is formed (~3%) is probably derived from the small amount of P420 present (i.e. Fig. 6) and is consistent with the low amount of heme transferred to apo-b₅ (Figs. 2 and 3).

Conclusions-- The literature contains reports of at least four P450s for which catalysis is stimulated by apo-b₅ as well as b₅ (22, 28-31). However, some P450s (e.g. 2E1) are clearly stimulated by b₅ but not by apo-b₅ (28, 66), consistent with a role for electron transfer. Recently Guryev et al. (39) have raised the possibility that the stimulations by apo-b₅ may be the result of transfer of heme from P450 preparations to apo-b₅ to form b₅, which then functions in an electron transfer role. The facile equilibration of P450 heme with the medium is highly unlikely, but the possibility exists that adventitiously bound heme could be transferred to fill the role of the b₅ prosthetic group. The varying effects of apo-b₅ versus b₅ on different P450s (e.g. 2E1 versus 3A4, 4A7, 17A, and 2C9) might be attributable to differences in the content of loosely bound heme, if this hypothesis has merit. To investigate these possibilities, we re-examined several features of the stimulation of some P450 3A4 catalytic activities by apo-b₅.

We confirm our previous results (22, 28) on the stimulation of P450 activities by b₅ and apo-b₅, using four different P450 3A4 preparations isolated with a different procedure. Apo-b₅ and b₅ are similar in their stimulation (Fig. 1), and all of the P450 3A4 preparations had nearly equivalent amounts of detectable P450 and total heme as judged by the application of the generally used extinction coefficients. Spectral studies indicate that some of these P450 3A4 preparations do contain trace heme that can be transferred to apo-b₅ (Fig. 3), but only to the extent of ~5%, which will not account for the observed concentration-dependent stimulation of P450 3A4 (Fig. 1). Highly purified apomyoglobin had no discernable effect on the stimulation of either testosterone 6-hydroxylation or nifedipine oxidation by b₅ or apo-b₅, even with a P450 preparation with a considerable amount of P420. A reconstituted heme oxygenase system had little effect on the P450 spectrum after incubation with P450 3A4 (Fig. 6), and only ~3% of the heme was converted to biliverdin (Fig. 7), ruling out the presence of appreciable adventitiously bound heme and the use of intact P450 3A4 as a substrate by heme oxygenase. A further important argument against the necessity of heme transfer to apo-b₅ for stimulation is an experiment done previously (22) on the lack of substitution of apo-b₅ in NADH-b₅ reductase/P450 3A4-dependent testosterone 6-hydroxylation, which was repeated here with the same result.

We do not fully understand the details of how apo-b₅ and, by extension, b₅ stimulate all P450 activities. This is not unusual in the context of modern biochemistry, where many protein interactions result in remarkable changes in biological activity but are not well understood. The complexity of the interactions of P450 3A4 with various ligands has been reviewed recently (67). Because P450 2E1 activities are stimulated by b₅ but not by apo-b₅ (66), a case can clearly be made there for electron transfer. With P450 3A4, both apo-b₅ and b₅ stimulated the reduction of ferric P450 in the presence of the substrate testosterone (22, 27); this effect was not observed with P450 2C9 (28) and was observed to only a limited extent with 4A7 (30, 31). The K_m (testosterone) and oxidation-reduction potential for P450 3A4 were not changed (22). Other studies with P450s 3A4, 17A, and 4A7 (22, 28, 29, 31) have not defined mechanisms of apo-b₅ stimulation to date, and these P450s may differ in the exact roles that b₅ and apo-b₅ play.

	FOOTNOTES

^* This research was supported in part by United States Public Health Service Grants R35 CA44353, R01 CA90426, and P30 ES00267 and grants from the Ministry of Education, Science, and Culture of Japan and the Ministry of Health and Welfare of Japan.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: Dept. of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Building (MRBI), 23^rd and Pierce Avenues, Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: guengerich@toxicology.mc.vanderbilt.edu.

Published, JBC Papers in Press, June 18, 2001, DOI 10.1074/jbc.M105011200

² H. Yamazaki, M. Nakamura, T. Komatsu, K. Ohyama, M. Nakamura, S. Asahi, N. Shimada, F. P. Guengerich, T. Shimada, M. Nakajima, and T. Yokoi, submitted for publication.

	ABBREVIATIONS

The abbreviations used are: P450, cytochrome P450 (also termed heme-thiolate protein P450 by the Enzyme Commission (EC 1.14.14.1) (1)); P420, cytochrome P420 (spectrally detected denatured forms of P450); b₅, cytochrome b₅ (EC 4.4.2 group); apo-b₅, b₅ devoid of heme; HPLC, high pressure liquid chromatography.

	REFERENCES

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