Structural Determinants of Active Site Binding Affinity and Metabolism by Cytochrome P450 BM-3

Archives of Biochemistry and Biophysics Vol. 387, No. 1, March 1, pp. 117–124, 2001 doi:10.1006/abbi.2000.2246, available online at http://www.idealibrary.com on

Structural Determinants of Active Site Binding Affinity and Metabolism by Cytochrome P450 BM-3 L. Ashley Cowart,* John R. Falck,† and Jorge H. Capdevila* ,‡ ,1 ‡Department of Medicine and *Department of Biochemistry, Vanderbilt University Medical, Nashville, Tennessee 37232; and †Department of Biochemistry, University of Texas Southwestern Medical School, Dallas, Texas 75235

Received September 26, 2000, and in revised form November 21, 2000; published online February 2, 2001

The determinants of the regio- and stereoselective oxidation of fatty acids by cytochrome P450 BM-3 were examined by mutagenesis of residues postulated to anchor the fatty acid or to determine its active site substrate-accessible volume. R47, Y51, and F87 were targeted separately and in combination in order to assess their contributions to arachidonic, palmitoleic, and lauric acid binding affinities, catalytic rates, and regio- and stereoselective oxidation. For all three fatty acids, mutation of the anchoring residues decreased substrate binding affinity and catalytic rates and, for lauric acid, caused a significant increase in the enzyme’s NADPH oxidase activity. These changes in catalytic efficiency were accompanied by decreases in the regioselectivity of oxygen insertion, suggesting an increased freedom of substrate movement within the active site of the mutant proteins. The formation of significant amounts of 19-hydroxy AA by the Y51A mutant and of 11,12-EET by the R47A/Y51A/F87V triple mutant, suggest that wild-type BM-3 shields these carbon atoms from the heme bound reactive oxygen by restricting the freedom of AA displacement along the substrate channel, and active site accessibility. These results indicate that binding affinity and catalytic turnover are fatty acid carbon-chain length dependent, and that the catalytic efficiency and the regioselectivity of fatty acid metabolism by BM-3 are determined by active site binding coordinates that control acceptor carbon orientation and proximity to the heme iron. © 2001 Academic Press Key Words: arachidonic acid; cytochrome P450; fatty acid monooxygenases; ␻-hydroxylase; epoxygenase.

1

To whom correspondence should be addressed. Vanderbilt University Medical School, Medical Center North S-3223, Nashville, TN 37232. 0003-9861/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

Cytochrome P450 BM-3 is a soluble cytochrome P450 isoform from Baccillus megaterium consisting of a heme domain fused to a flavoprotein reductase domain (1– 4). Despite its bacterial origin, BM-3 has structural and functional homology to mammalian fatty acid hydroxylases of the P450 4A gene subfamily (4) and it is often used as a model for mammalian P450s (5, 6). BM-3 oxidizes arachidonic acid (AA) to 18(R)-hydroxyeicosatetraenoic acid and 14(R),15(S)-epoxyeicosatrienoic acid (ⱖ95% optical purity; 80 and 20% of total metabolites, respectively) with turnover in the thousands (7, 8). This high degree of regio- and stereoselectivity are unusual for P450 catalyzed oxidations of unbiased, acyclic molecules such as AA, and consequently the protein structural determinants of AA regio- and stereoselective oxidation by this protein are of special interest. The crystal structure of the heme domain of BM-3 at 2 Å resolution revealed a protein in the shape of a triangular prism, consisting of distinct amino and carboxy-terminal domains (the ␣- and ␤-domains, respectively) with the heme prosthetic group nestled between them (9). A long, narrow channel leading from the surface of the ␤-domain to the heme was defined as the substrate access channel (9). This channel is more or less cylindrical in shape, is lined with mostly nonaromatic hydrophobic residues, and has an average width of 8 to 10 Å (9). Within Van der Waals radius of the heme is F87, which is hypothesized to control heme access and to serve as a determinant of the regioselectivity of AA metabolism by controlling the degree of substrate lateral mobility along the longitudinal axis of the channel (7, 8). Replacement of F87 with V yields a mutant protein that catalyzes only AA 14,15-epoxidation (8), and replacement with A yields a lauric (C12) and myristic (C14) acid ␻-hydroxylase, although in these last two cases product identification was based 117

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on NMR analysis, without further structural documentation (10). R47 is located at the surface opening of the access channel with its charged guanidinium side chain protruding into the lumen of the channel (9, 11). This property of R47 led to the hypothesis that it stabilizes substrate binding by ion-pairing with the fatty acid carboxylate moiety (9). Replacement of R47 with A decreased the binding affinity and catalytic activity of the enzyme towards AA but it had only moderate effects on the regioselectivity of oxygen insertion (8), suggesting that additional protein residues contributed to substrate anchoring and active site binding stabilization. The crystal structure of palmitoleic acid (PA) bound BM-3 provided a fresh perspective on amino acid-fatty acid carboxylate interactions and their role in substrate binding stabilization (11) and showed that, in addition to R47 charge-pairing, the hydroxyl of Y51 was within hydrogen bonding distance of the fatty acid carboxylate (11). This atomic structure revealed a protein conformation more closed than that of the substrate-free protein (9, 11), and indicated that a reduction of the distance between the ␣- and ␤-domains was required for PA oxidation, since the acceptor carbon was ⬃8 Å away from the heme (11). The experiments suggested reduction with Na 2S 2O 4, moved substrate carbon acceptor within oxygen transfer distance from the heme iron (⬃4 Å) (12). Furthermore, additional indirect approaches have supported the notion that conformational changes do occur throughout the catalytic cycle of BM-3 (13–15). To identify determinants of BM-3 regioselectivity during AA catalysis, and to probe the role that the structural characteristics of the substrate play in active site binding and catalytic activity, we have targeted for site-directed mutagenesis residues R47, Y51, and F87 of BM-3, either alone or in combination, and compared their effects on the metabolism of fatty acids of different lengths and degrees of saturation. The results indicate that all three residues participate in substrate binding and contribute to regioselective fatty acid metabolism, and that their relative importance is fatty acid specific and carbon chain length-dependent. Furthermore, we provide evidence to indicate that the conformations of the enzyme–substrate complexes during catalytic turnover are multiple and more or less substrate specific. MATERIALS AND METHODS Cytochrome P450 BM-3 as well as the R47A and F87V mutant cDNAs in pIBI were gifts from Dr. Julian Peterson (University of Texas Southwestern Medical School, Dallas, TX). Radiolabeled fatty acids were from American Radiolabeled Chemicals, Inc. (Saint Louis, Missouri) and unlabeled fatty acids were from NuCheck Prep, Inc. (Elysian, Minnesota) except sodium laurate, which was from Sigma (St. Louis, Missouri).

cDNA cloning, expression, and protein purification. cDNAs coding for the Y51A, and R47A/Y51A mutants were generated by PCR of a 1014-base pair fragment using upstream primers encoding the desired mutations and the appropriate template (BM-3 cDNA for the Y51A mutant, and the Y51A cDNA for the R47A/Y51A mutant). PCR products were cloned into the pCR 2.1 vector and, after sequence analysis, an AgeI/MfeI fragment was isolated, purified, and subcloned into the pCW-BM3 expression vector using compatible restriction sites. The R47A/Y51A/F87V mutant was generated by subcloning a 3962 bp AflII/AvaI fragment isolated from the pCW BM-3 F87V mutant into the pCW BM-3 R47A/Y51A. Prior to expression, cloning site integrities were confirmed by sequence analysis. cDNAs coding for wild type and mutant P450 BM-3 isoforms were expressed using DH5␣ Escherichia coli grown in modified TB medium containing 1 mM ␦-aminolevulinic acid as described (7). After 44 h at 30°C, the cells were collected by centrifugation and the proteins extracted and purified as described (16). Purified proteins were dialyzed for 15 h versus 0.1 M potassium phosphate buffer pH 7.4 containing 10 ⫺5 M DTT and stored at ⫺80°C. Samples were discarded after one cycle of freezing and thawing. Spectral and enzymatic characterization. Difference spectra were determined in 0.1 M phosphate buffer pH 7.4 at 25°C. Fatty acids were dissolved in 0.1 M Tris–Cl, pH 8.0, and added incrementally with a Hamilton syringe such that total added volume was less than 1% of the sample volume. Spectral changes from 350 to 500 nm were monitored and K s values were calculated from double reciprocal plots of substrate concentration versus changes in absorbance between 387 and 420 nm. Enzyme incubations were performed exactly as described (7). Fatty acids were added as sodium salts in 0.1 M Tris–Cl, pH 8.0, to a final concentration of 100 ␮M. Hydrogen Peroxide formation was determined by the method of Hildebrandt et al. (17). Hydrogen peroxide formation was calculated from the absorbance difference observed between reactions performed in the presence or absence of catalase (30 U/ml catalase, Sigma, St. Louis, MO). Product characterization. AA metabolites were resolved and characterized by a combination of RP-HPLC and NP-HPLC as described (18). The chirality of 14,15-EET was determined as described (19). 18-hydroxyeicosatetraenoic acid was converted to the corresponding Mo¨sher ester and the enantiomers resolved by HPLC on a Chiralcel OD column (20). PA metabolites were analyzed by RPHPLC using the solvent system described in reference 18, and tentatively separated into epoxide and mono-hydroxyalcohols based on relative retention times. The less polar material (R t between 18 and 20 min) was tentatively identified as the PA 9,10-epoxide based on: (a) hydrogenation over PtO 2 yielded a molecule with a RP-HPLC retention time identical to that of the parent molecule, showing lack of an olefinic bond between carbons 9 and 10, and (b). Treatment with 9 M acetic acid (12 h, 22°C) changed the RP-HPLC of the molecule (R t: 7 min) showing an increased polarity, indicating acid catalyzed H 2O addition across the oxirane ring. The polar metabolites (R t between 11 and 13 min) were tentatively identified as hydroxy-PA based on: (a) catalytic hydrogenation over PtO 2 changed their RP-HPLC R t to 15.5 min, suggesting the presence of a 9,10 olefin in the parent molecule, (b) Treatment with 9 M acetic acid as above, had no significant effects on their RP-HPLC retention times, and (c) reaction with bis(trimetylsilyl)trifluoroacetamide (18) and pentafluorobenzylbromine (18), followed by NICI/GC/MS analysis (18) yielded an abundant ion fragment at m/z 341 (base peak), corresponding to the molecular weight of the [M-PFB] ⫺ ion fragment originating from the trimethylsilyl-ether-pentaflorobenzyl ester derivative of hexadecenoic acid.

RESULTS AND DISCUSSION

BM-3 has been chosen by us and others (5– 8) to model mammalian P450 fatty acid hydroxylases because: (a) it is functionally and structurally homolo-

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FATTY ACID BINDING AND OXIDATION BY P450 BM-3 TABLE I

Spectral Dissociation Constants for Binding of Arachidonic Acid to P450 BM-3 and Its Mutant Isoforms K s (␮M) wt R47A* Y51A F87V* R47A/Y51A R47A/Y51A/F87V

3⫾1 11 ⫾ 3 12 ⫾ 1 2⫾1 14 ⫾ 2 81 ⫾ 3

Note. Binding experiments were performed as described under Materials and Methods. Values are averages ⫾ SE (n ⬎ 3). * From Ref. (8).

gous to the members of P450 4A gene subfamily, and (b) the atomic structures of its substrate free and substrate bound forms are published and allowed the identification of putative substrate binding residues (9, 11). We have utilized these structures and targeted amino acid replacement techniques to study the role that three of these residues play in the binding and metabolism of AA by BM3, and compared these results to those obtained using PA and lauric acid (LA), two fatty acids for which there is published information (21, 22). In addition to being one of the best substrates ever reported for an NADPH-dependent, P450 catalyzed monooxygenation reaction (7), AA and its oxidation products have an extensive list of important physiological roles (23), and access to active regio- and stereoselective AA monooxygenases is of current interest. Spectroscopic analysis of substrate binding. Determination of substrate induced spectral perturbations allows examination of substrate interactions within the heme environment that are independent of electron transfer and redox changes. As reported (7, 8) and suggested by K s values in the low ␮M range (Table I), AA binds to BM3 with high affinity. Replacement of either of the proposed substrate anchoring residues (R47 or Y51) (11) for alanine led to increases in K s values and decreased binding affinity for AA (Table I). Interestingly, the replacement of both R47 and Y51 with alanine did not result in further changes in AA binding affinity, and yielded a mutant protein with a K s value for AA similar to that of the single mutants (Table I). The BM-3 atomic structure indicated that the aromatic side chain of F87, positioned immediately above and perpendicular to the plane of the heme prosthetic group, constricts the access channel volume available to the substrate and thus, controls heme access (7–9). Replacement of F87 with A or V markedly changed the regioselectivity of BM-3 for AA and LA oxidation (8, 10), but had little or no effect on the fatty acids K s values and thus, binding affinity (8, 10). These studies

showed that the regiochemistry of oxygen insertion and thus, the relative spatial relationship between the heme-oxygen donor and the substrate carbon acceptor was independent of binding affinity. A marked decrease in the affinity of BM3 for AA was observed when the three targeted residues (i.e., R47, Y51, and F87), were replaced at the same time thus, as shown in Table I, the triple mutant (R47A/Y51A/F87V) binds AA with a K s value significantly higher than that of the wild type or any of the other mutants. These results suggest that as a group, R47, Y51, and F87, play an important role in AA binding and that their coordinated contributions are required for a high affinity binding of AA to the BM3 active site. A caveat to this interpretation is that, as with most site-directed mutagenesis studies, the overall structural consequences of these mutations remain unknown. The contributions of the substrate carbon chain length and saturation, as well as the role of the targeted protein residues to substrate binding affinity were further explored using PA and LA. Replacement of Y51 with A, either singly or in conjunction with R47, abolished the spectrally detectable interactions of PA with BM-3. Thus, compared to AA, Y51 plays a decisive role in stabilizing the binding of PA to the active site of BM3, suggesting that the anchoring role proposed for this residue (11) is likely to be substrate carbon chain length-dependent. Finally, replacement of F87 with V in the R47A/Y51A mutant partially restored PA binding to the protein although the high K s values indicate the limited binding affinity of the triple mutant for PA (Table II). Our efforts to characterize the binding of LA to BM3 or its mutant isoforms were unsuccessful. Spectral titrations with sodium laurate yielded poorly defined spectra lacking defined isosbestic points, and a proper relationship between LA concentration and typical type I binding spectra could not be observed at fatty acid concentrations within its limit of solubility (Fig. 1). While the K s values reported for the binding of LA to BM-3 vary from ⬃30 to 840 ␮M (24 –27), the limited water solubility of this fatty acid and the reported high K s values complicate the interpretation of LA binding affinities using spectral methods. TABLE II

Spectral Dissociation Constants for Binding of Palmitoleic Acid to P450 BM-3 and Its Mutant Isoforms K s (␮M) wt R47A F87V R47A/Y51A/F87V

7⫾1 34 ⫾ 5 6⫾2 105 ⫾ 3

Note. Binding experiments were performed as described under Materials and Methods. Values are averages ⫾ SE (n ⬎ 3).

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COWART, FALCK, AND CAPDEVILA TABLE III

Arachidonic Acid Oxidation by BM-3 and Its Mutant Isoforms

wt R47A Y51A F87V R47A/Y51A R47A/Y51A/F87V

min ⫺1

Percentage of wt rate

2330 ⫾ 185 1824 ⫾ 34 563 ⫾ 54 948 ⫾ 50 235 ⫾ 20 493 ⫾ 54

100 78 24 41 10 21

Note. Incubations were performed as described under Materials and Methods, and rates were estimated by determination of product formed within the first 30 s after addition of NADPH. See Results and Discussion for details. Values are averages ⫾ SE (n ⬎ 3).

FIG. 1. Spectral analysis of the interactions of the Y51A BM-3 mutant with arachidonic and lauric acids. The fatty acids were added to a 1 ␮M solution of P450 in either 0.4 (AA) or 8 ␮M (LA) increments. (A) AA titration (0 –2.8 ␮M). (B) LA titration (0 –16 ␮M). See Materials and Methods for more details.

The results discussed above indicate that fatty acid binding by BM3 involves complex molecular interactions between distinct amino acid residues that control substrate anchoring (R47 and Y51) and channel volume (F87), as well as yet to be characterized substratedependent interactions between the fatty acid C-H backbone and the residues lining the surface of the access channel (9). Furthermore, our data indicate that the relative importance of these interactions are substrate dependent and that their generality may be limited. Thus, it seems that for PA, the major contributors to binding affinity are interactions with the putative anchoring groups (i.e., Y51 and R47), while for AA additional contributions are provided by its nearly perfect fit into the volume of the substrate access channel (7). In contrast, PA shows a less than perfect fit, with its acceptor carbon ⬃8 Å from the heme iron (11).

Enzymatic characterization. Inasmuch as metabolism by P450 requires, in addition to proper binding affinity and geometry, the redox-coupled activation of molecular oxygen, the delivery of an active form of oxygen to a properly positioned C-H acceptor, and product release, determinations of catalytic rates and product regio- and stereochemical properties can provide accurate, although indirect, descriptions of substrate binding rigidity and orientation within the spatial coordinates of the protein active site. Exceedingly high rates and limited solubility have complicated the determination of initial velocities during the analysis of AA metabolism by BM-3 (7, 8), and HPLC-based measurements of product formed within the first 30 s of the incubation have been previously utilized as estimates of the rate of AA oxidation by BM3 (7, 8). As reported (7, 8, 28) and shown in Tables III and IV, AA and PA are metabolized by BM-3 at rates approaching the rates of electron transfer from NADPH to the BM3 heme iron (29). In contrast, LA is metabolized by BM3 at a fraction of the rates observed with either AA or PA (3– 4% of the AA or LA rates) (Tables II, IV, and V). The

TABLE IV

Palmitoleic Acid Oxidation by BM-3 and Its Mutant Isoforms

wt R47A Y51A F87V R47A/Y51A R47A/Y51A/F87V

min ⫺1

Percentage of wt rate

2396 ⫾ 185 1690 ⫾ 27 516 ⫾ 81 1869 ⫾ 70 76 ⫾ 8 348 ⫾ 16

100 71 22 78 3 15

Note. Incubations were performed as described under Materials and Methods, and reaction rates were estimated by determination of product formed within the first 30 s after addition of NADPH. See Results and Discussion for details. Values are averages ⫾ SE (n ⬎ 3).

FATTY ACID BINDING AND OXIDATION BY P450 BM-3 TABLE V

Lauric Acid Oxidation by BM-3 and Its Mutant Isoforms

wt R47A Y51A F87V R47A/Y51A R47A/Y51A/F87V

min ⫺1

Percentage of wt rate

81.1 ⫾ 2.7 95.2 ⫾ 14.8 26.7 ⫾ 0.9 78.8 ⫾ 17.0 5.7 ⫾ 0.14 5.2 ⫾ 1.2

100 117 33 97 7 6

Note. Incubations were performed as described under Materials and Methods, and reaction rates were estimated by determination of product formed within the first 30 s after addition of NADPH. See Results and Discussion for details. Values are averages ⫾ SE (n ⬎ 3).

values in Table V are based on HPLC-based measurement of product formation and are similar to those previously reported using a similar method (21); however, they are substantially lower than published estimates of product formation using indirect methods (2, 12, 16, 24, 30). Replacement of the proposed hydrogen bonding donor Y51 with alanine caused substrate-independent decreases in the rates of fatty acid metabolism (24, 22, and 33% of wt for AA, PA, and LA, respectively) (Tables III–V). In contrast, replacement of the proposed ionpairing R47 with A had only a minor negative impact on AA and PA reaction rates (78 and 71% of wt rates, respectively) (Tables III and IV) and led to a slight stimulation of LA turnover (Table V). On the other hand, replacement of the proposed heme gating residue, F87, with A did not affect the rates of LA oxidation (Table V), but had carbon chain length-dependent effects on PA and AA metabolism (78 and 41%, of wt rates, respectively) (Tables III and IV). Comparison of the data in Tables I–V indicates that: (a) the effects of replacing R47 with A on binding properties and reaction rates are limited, more or less similar, and substrate-dependent, and (b) overall, the consequences of replacing Y51 with A are more pronounced than those resulting from R47 modifications (Tables I–V). Furthermore, while mutations at Y51 caused carbon length-dependent decreases in binding affinity (Tables I and II), their effects on rate were substrate-independent (Tables III–V). Paradoxically, replacement of Y51 with A significantly reduced the spectral manifestation of PA binding (Table II), but the Y51A mutant retained robust PA metabolic activity (Table IV). We conclude that: (a) Y51 is essential for full catalytic activity and that its role in catalytic turnover is independent of the substrate carbon chain length, (b) in contrast, R47 play its most important, carbon chain-independent, role during substrate binding stabilization, but it has a more limited role during catalytic turnover, and (c) F87 plays an important role

121

in determining the volume of the substrate access channel that is available to the substrate. Its effects are more marked the longer the fatty acid carbon chain. The atomic structures of substrate free and bound BM3 show that R47 and Y51 are located in the protein’s ␤1–2 loop, an area of high conformational flexibility and well suited for their proposed role in anchoring fatty acid carboxylates during catalytic turnover (9, 11). In agreement with this, simultaneous replacement of R47 and Y51 with A yielded a mutant protein nearly inactive towards AA, PA, or LA (Tables III–V). These results indicate that these residues contribute to position the substrate in the proper orientation during the protein conformational changes proposed to occur during heme iron reduction and catalytic turnover (12–15). Finally, replacement of F87 in the double mutant to generate the R47A/Y51A/F87V mutant increased the turnover rate for AA and PA, but not for LA, again reflecting its proposed substrate carbon-length dependent role in heme gating (8). In summary, the effects described indicate that the residues targeted for replacement control mostly binding rigidity and the relative distance between the substrate carbon acceptor and the heme-bound oxidant; as opposed to the chemistry of either the reactive oxygen or acceptor atom(s). While for many P450 enzymes substrate binding is not generally thought to directly limit rates of catalytic turnover (32), recent work with BM-3 has suggested a relationship between LA binding and the efficiency of electron transfer between the BM-3 reductase FAD and FMN cofactors and the heme iron of substrate-bound BM-3 (33, 34). Since changes in binding affinity and geometry could affect not only productive turnover but also the degree of coupling between NADPH utilization, oxygen reduction, and product formation (35), we investigated this issue by measuring rates of H 2O 2 formation during fatty acid metabolism. Hydrogen peroxide formation. The generation of variable amounts of H 2O 2 during catalytic turnover by several microsomal P450s is well documented (35). This “uncoupling” of NADPH oxidation from oxidative metabolism results from the diversion of NADPH-derived reducing equivalents towards the reduction of oxygen and their release as H 2O 2 (35). H 2O 2 can be formed by either dismutation of the oxy–P450 complex or by its one electron reduction (35). As reported (7), AA metabolism by BM-3 does not generate H 2O 2 and, furthermore, regardless of the nature or number of replacements introduced, AA metabolism by the different BM3 mutant forms remained fully coupled and hydrogen peroxide was not a reaction product. In contrast, and with the sole exception of the Y51A mutant, LA metabolism by BM-3 and its mutant isoforms re-

122

COWART, FALCK, AND CAPDEVILA TABLE VI

Formation of Hydrogen Peroxide During Incubation of BM-3 or Its Mutant Isoforms with Lauric Acid Lauric acid Hydrogen metabolites peroxide (mol/mol/min) (mol/mol/min) Total Uncoupling wt R47A Y51A F87V R47A/Y51A R47A/Y51A/F87V

81 95 27 79 6 5

27 81 N.D. 72 11 82

108 176 27 150 17 87

0.25 0.46 — 0.48 0.66 0.94

Note. Rates of hydrogen peroxide formation were determined as described under Materials and Methods. Hydrogen peroxide values are represented as the difference between total ferrous ammonium sulfate reactive material and catalase sensitive ferrous ammonium sulfate reactive material. Uncoupling is expressed as the ratio of mol peroxide formed/(mol peroxide formed ⫹ mol oxidized fatty acid formed). Values are averages (n ⫽ 3, SE ⬍ 10%).

sulted in variable degrees of uncoupling and significant hydrogen peroxide formation (Table VI). These results, in conjunction with its low rates of metabolism (Table VI), indicate that productive turnover occurs with LA occupying a less than optimal spatial orientation within the active site. NMR data has indicated that the hydroxylation of short chain fatty acids by BM-3 requires redox-dependent conformational changes to bring the acceptor carbon within hydroxylation distance from the heme (10, 12). Of interest, replacement of R47 with A or of F87 with V had similar effects on the rates of H 2O 2 generation by the resulting mutants (Table VI). Thus, while the rates of LA metabolism were not significantly altered, these replacements increased electron flow thorough the LA-mutant BM-3 catalytic complexes and H 2O 2 formation more than doubled (Table VI). On the other hand, replacement of Y51 with A either singly or in combination with R47, eliminated (Y51A mutant) or drastically reduced (R47A/Y51A mutant) H 2O 2 generation (Table VI) and LA metabolism (Table V). Finally, the triple R47/Y51/ F87V mutant generated H 2O 2 as nearly the sole product of redox hemoprotein turnover and functioned as an LA-dependent NADPH oxidase instead of a LA monooxygenase (Table VI). Importantly, NADPH-dependent H 2O 2 formation by BM-3 and its mutant isoforms required the presence of LA (not shown). It is apparent from the above that the levels of NADPH-dependent H 2O 2 formation by BM-3 are substrate carbon chain length-dependent. Furthermore, we conclude that: (a) the partition ratio between rates of product and H 2O 2 formation, i.e., the degree of uncoupling, is determined predominantly by (i) the proximity of the acceptor carbon to the heme bound oxidant, and (ii) the rigidity with which the protein con-

trols the fatty acid binding coordinates within the volume of the access channel. Heme proximity and rigid binding favor metabolism (as with AA); increased freedom of movement and distance from the heme favor H 2O 2 generation (as with LA), (b) Interactions between Y51 and the LA carboxylate moiety are the major contributors to binding rigidity, while R47 and F87 play secondary roles in controlling the freedom of displacement of LA within the volume of the BM-3 access channel, and (c) an overall poor fit between the volume of the LA molecule and the volume of the BM-3 access channel, as well as anchoring of the fatty acid at or near the outer limits of active site heme-iron-oxidant transfer sphere provide the rationale for the fact that LA is comparatively a very poor substrate for BM-3. It is interesting to notice that, with the sole exception of the Y51A mutant, measurements of NADPH or O 2 consumption are poor indicators of LA metabolism (Table V). Regio- and stereoselectivity of oxygenation. The atomic structure and the analysis of the regio- and stereochemistry of oxygenation by BM-3 have been utilized to estimate substrate binding coordinates and model fatty acid binding to the enzyme’s active site (7, 8). Thus, for example, replacement of F87 with V converted BM-3 from a predominantly AA ␻-2 hydroxylase into a regio- and stereoselective 14(S),15(R) AA epoxygenase (8); an effect attributed to the removal of the F87-side chain steric barrier and increased access of the 14,15-olefin to heme iron (8). Based on these studies, as well as the binding and metabolism data in Tables I–VI, we hypothesized that (a) weakening AA anchoring at the mouth of the channel should increase the substrate’s freedom of movement along the longiditunal axis of the access channel, and facilitate hydroxylation at/or proximal to the AA ␻-carbon, and (b) that the combined effects of relaxing carboxylate anchoring and increasing heme access should facilitates access of the AA 11,12-olefin to the protein oxygenating locus. Replacement of Y51 with A, either singly or in combination with R47 did result in metabolism at the AA ␻-1 carbon and generated significant amounts of 19-hydroxyeicosatetraenoic acid, a new product of AA oxygenation by BM-3 (Table VII). Compared to BM-3 or the Y51A protein, the Y51A/R47A double mutation leads to significant reductions in AA binding affinity and rate of metabolism, as well as in decreased regioselectivity of oxygenation (Tables I, III, and VII). These results support crystallographic data suggesting that these two residues may work as a functional unit during substrate anchoring (11, 36). As shown in Table VII, the formation of 19-hydroxy-AA by the R47/Y51A double mutant was associated with significant increases in 14,15-epoxygenase activity (Table VII); further supporting the idea that in wild-type BM-3, these

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FATTY ACID BINDING AND OXIDATION BY P450 BM-3

residues limit the freedom of AA displacement along the longiditunal axis of the access channel (9, 10). Indeed, increasing the substrate accessible volume by replacing F87 with V in the Y51A/R47A mutant removed the F87-heme steric barrier and led to an increased rate of AA metabolism and formation of small but significant levels of the 11,12-epoxide of AA (Table VII), an olefin fully shielded from the reactive oxygen in BM-3 or its F87V mutant (7, 8). Based on an NMR analysis, it was concluded that replacement of F87 with A made the ␻-carbon of LA accessible to hydroxylation by BM-3 (10); however, under our experimental conditions we failed to obtain either chromatographic or GC/MS evidence for the formation of 12-hydroxy-LA by wild-type BM-3 or any of its mutant isoforms. Importantly, and as shown in Table VIII, the above described changes in the rates and regioselectivity of AA metabolism occurred with preservation of the overall enantiofacial selectivity of oxygen insertion by the BM-3 mutant isoforms, further corroborating the proposal that the targeted residues control binding rigidity and accessible volume but not the orientation of the acceptor carbons with regards to the spatial coordinates of the active site and the heme bound oxidant. Early work by A. Fulco and collaborators demonstrated that the partition ratio between PA epoxidation and hydroxylation was pH-dependent, i.e., acidic pHs favored PA epoxidation over hydroxylation. These pioneering studies suggested that the fatty acid carboxylate played a critical role in determining catalytic outcome (22). More recently, these early observations have been attributed to changes in the substrate anchoring properties of R47 and Y51 due to pH-induced increases in fatty acid protonation (36). Our studies of the effects of replacing these residues with A on PA epoxidation and hydroxylation confirmed the above predictions. Compared to BM-3, the R47A/Y51A mutant isoform favored PA epoxidation over hydroxylation (1.9 and 3.5 nmol of epoxide formed/nmol of total hydroxylated TABLE VII

Regioselectivity of Arachidonic Acid Oxidation by BM-3 and Its Mutant Isoforms Percentage of total metabolites

19-OH 18-OH 14,15-EET 11,12-EET

wt

Y51A

R47A/Y51A

R47A/Y51A/F87V

N.D. 81 19 N.D.

16 74 10 N.D.

11 49 40 N.D.

N.D. N.D. 96 4

TABLE VIII

Stereochemical Analysis of Arachidonic Acid-Derived Metabolites of BM-3 and Its Mutant Isoforms 18-OH

wt R47A Y51A R47A/Y51A F87V R47A/Y51A/F87V

14,15-EET

R (%)

S (%)

S, R (%)

R, S (%)

96 95 99 86 N.D. N.D.

4 5 1 14 N.D. N.D.

99 99 97 99 99 99

1 1 3 1 1 1

Note. Determined as described under Materials and Methods. N.D., not detectable.

products, for BM-3 and the R47/Y51 mutant protein, respectively, data not shown). Finally, as with AA, replacement of F87 with V in either BM-3 or the R47A/ Y51A mutant converted both enzymes into regioselective PA 9,10-epoxygenases (⬃100% of total recovered products, data not shown). From the results summarized, we conclude that: (a) substrate binding rigidity and acceptor carbon orientation, as opposed to oxygen chemistries, are major determinants of overall reaction rates, regio- and stereoselectivity of oxygenation, and H 2O 2 formation; (b) Both R47 and Y51 participate in anchoring the fatty acid carboxylate at the mouth of the access channel. The contribution of these residues to binding affinity and rigidity is largely determined by the length of the fatty acid carbon chain; (c) F87 plays an important role in determining catalytic outcomes by controlling the volume of the heme environment accessible to the substrate. This role is more significant the longer the carbon chain length of the substrate; (d) a significant, substrate-specific contribution to binding affinity is derived from yet to be determined interactions between the substrate carbon– hydrogen backbone and the side chains of residues lining the entire length of the protein’s access channel. The almost perfect fit between AA and the BM-3 binding volume (7) allows for the fast regioselective and highly asymmetric oxidation of this fatty acid. ACKNOWLEDGMENTS This work was supported by USPHS-NIGM Grants 31278 (J.R.F.) and 37922 (J.C.H.).

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