The Catalytic Site of Cytochrome P4504A11 (CYP4A11) and Its L131F Mutant*

CYP4A11, the principal known human fatty acid ω-hydroxylase, has been expressed as a polyhistidine-tagged protein and purified to homogeneity. Based on an alignment with P450BM-3, the CYP4A11 L131F mutant has been constructed and similarly expressed. The two proteins are spectroscopically indistinguishable, but wild-type CYP4A11 primarily catalyzes ω-hydroxylation, and the L131F mutant only ω-1 hydroxylation, of lauric acid. The L131F mutant is highly uncoupled in that it slowly (ω-1)-hydroxylates lauric acid yet consumes NADPH at approximately the same rate as the wild-type enzyme. Wild-type CYP4A11 is inactivated by 1-aminobenzotriazole under turnover conditions but the L131F mutant is not. This observation, in conjunction with the binding affinities of substituted imidazoles for the two proteins, indicates that the L131F mutation decreases access of exogenous substrates to the heme site. Leu-131 thus plays a key role in controlling the regioselectivity of substrate hydroxylation and the extent of coupled versus uncoupled enzyme turnover. A further important finding is that the substituted imidazoles bind more weakly to CYP4A11 and its L131F mutant when these proteins are reduced by NADPH-cytochrome P450 reductase than by dithionite. This finding suggests that the ferric enzyme undergoes a conformational change that depends on both reduction of the iron and the presence of cytochrome P450 reductase and NADPH.

CYP4A11, one of the three known members of the human CYP4 family (1)(2)(3)(4), is the only established fatty acid -hydroxylase in human liver and kidney (5,6). Little is actually known about the structure and function of CYP4A11 other than its fatty acid hydroxylation activity and its requirement for both cytochrome P450 reductase and cytochrome b 5 for optimal activity (2). In the presence of these electron transfer partners, CYP4A11 catalyzes the -hydroxylation of lauric, palmitic, and arachidonic acids with turnover numbers of 9.8, 2.2, and 0.6 min Ϫ1 , but it does not hydroxylate prostaglandins (2). The paucity of information on this enzyme is unfortunate, because current evidence suggests that it may play an important role in human physiology and may be a potential drug target. These conclusions derive from studies with rats which show that (a) 20-hydroxyeicosatetraenoic acid, the -hydroxylation product of arachidonic acid, is a potent vasoconstrictor (7,8), (b) 20hydroxyeicosatetraenoic acid is a major arachidonic acid metabolite in the spontaneously hypertensive rat (9), and (c) inhibition of arachidonic acid -hydroxylation by 17-octadecynoic acid alters renal function (10).
Most of our understanding of CYP4A11 derives from an extrapolation of the available information on rat CYP4A1, with which it exhibits 76% sequence identity (1). A defining feature of the CYP4A enzymes is their ability to favorover (-1)hydroxylation, a preference that requires the thermodynamically disfavored breaking of a strong primary terminal methyl C-H bond rather than a weaker secondary C-H bond of the -1 (or -n, n Ͼ 0) methylene group (11). In fact, the default regiospecificity for cytochrome P450 enzymes not specifically evolved as -hydroxylation catalysts is (-1)-rather than -hydroxylation (12)(13)(14). The mechanism by which the -hydroxylation regiospecificity is enforced is not known, although we recently reported that the CYP4A1 specificity is partially shifted toward (-1)-hydroxylation by a D323E mutation in the I-helix of the protein (15). Mutation of this residue was based on a sequence alignment of CYP4A1 with P450 BM-3 (CYP102).
In the absence of a crystal structure for a eukaryotic, membrane-bound P450 enzyme inferences about the structure of the CYP4A active site must be drawn from the crystal structures of four soluble bacterial enzymes: P450 cam (CYP101) (16), P450 terp (CYP108) (17), P450 BM-3 (18), and P450 eryF (CYP107) (19). Of particular relevance here is P450 BM-3 , a bacterial protein in which a P450 heme 1 domain is fused to a P450 reductase-like flavoprotein domain. The heme domain exhibits ϳ30% similarity to the CYP4A enzymes and the flavoprotein domain ϳ30 -35% similarity to the mammalian P450 reductases (20). P450 BM-3 also catalyzes fatty acid hydroxylation but at the -1, -2, and -3 rather than positions (21). The fatty acid in a crystal structure of substrate-bound P450 BM-3 lies in a long channel with the carboxylic acid group hydrogen bonded to Tyr-51 and Arg-47 near the channel entrance (22). The hydrocarbon terminus binds near the heme, but access of the terminal methyl to the heme iron atom is sterically hindered by Phe-87 (22). Anchoring the two ends of the fatty acid at the ends of a relatively wide substrate binding channel allows the enzyme to bind and regioselectively oxidize fatty acids of different chain lengths. Mutation of Phe-87 to a smaller alanine residue largely shifts the enzymatic regiospecificity from (-1/ 2/3)-to -hydroxylation (23). Unfortunately, the structural alterations responsible for this mutation-dependent shift in re-giospecificity are obscured by the finding through NMR studies that the enzyme-substrate complex undergoes a major conformational change when the iron is reduced from the ferric to the ferrous state (24). This conformational change is reported to shift the position of the substrate relative to the iron by a distance of 6 Å.
Only limited information is available on ligand or substrate binding to CYP4A11, but the specificity of CYP4A1 suggests that it binds substrates and ligands in a manner reminiscent of that attributed to P450 BM-3 . Thus, anchoring of the two fatty acid terminii is consistent with the finding that the binding affinity of fatty acid analogues with a terminal imidazole group is chain length-dependent and that this length dependence is suppressed when the carboxyl group is replaced by a nonpolar moiety (25). Furthermore, indirect evidence suggests both that the CYP4A1 active site is sterically constrained and that its -specificity is promoted by restricted access of the fatty acid chain to the oxidizing species (11). The steric factors controlling the reaction specificity may be complex, however, because CYP4A1 tolerates significant steric bulk near the hydroxylated carbon, as illustrated by the -hydroxylation of a fatty acid with a terminal tert-butyl group (26). Nevertheless, despite the sequence and functional similarities between CYP4A11 and CYP4A1 and their mutual relationship to P450 BM-3 , important differences exist between the two CYP4A proteins.
We report here expression and purification of poly(His)tagged CYP4A11 and its L131F mutant and a study of their catalytic and ligand binding properties. The results (a) indicate that Leu-131, which corresponds to Phe-87 in P450 BM-3 , plays an important role in determining the hydroxylation regioselectivity, (b) provide evidence that CYP4A11 undergoes a P450 reductase-dependent conformational change on reduction of the heme iron atom, (c) unmask important differences in the susceptibility of CYP4A11 and CYP4A1 to inhibitors, and (d) strengthen the use of P450 BM-3 as a model for the CYP4A family of proteins.
General Methods-Human NADPH-P450 reductase and cytochrome b 5 were expressed and purified according to the protocol outlined previously (15,28). The -hydroxylation activity and NADPH consumption rate of both CYP4A11 and its L131F mutant were determined as described previously for CYP4A1 (15). The reactions of CYP4A11 and L131F with phenyldiazene and 1-ABT were carried out as reported previously (15).
Expression of CYP4A11 and L131F-The pCWori plasmid containing either CYP4A11 or L131F was transformed into Escherichia coli XL-1 Blue (Stratagene), and a single colony was grown overnight in 2 ϫ YT medium (2 ϫ YT: 16 g of bacto-tryptone, 10 g of bacto-yeast, 5 g of NaCl in 1 liter total volume) with 200 g of ampicillin/ml. One liter of terrific broth containing 200 mg/liter ampicillin was inoculated with 2 ml of the saturated overnight culture and grown at 37°C to an A 600 of 0.5 to 0.6. At this point, 80 mg/liter ␦-aminolevulinic acid and 1 mM isopropyl-␤-D-thiogalactopyranoside were added. The flasks were cooled to 28°C and incubated an additional 24 h.
Purification of CYP4A11 and L131F-The cells were harvested by centrifugation at 4000 ϫ g for 20 min and were then frozen at Ϫ70°C for storage overnight. The cells were then thawed and resuspended in 100 mM Tris (pH 7.8) containing 1 mM EDTA and 20% glycerol. The suspension was stirred with lysozyme for 1 h at 4°C. Then 10 mM MgCl 2 and DNase I were added, and the suspension was stirred an additional 30 min at 4°C. Cytosolic proteins were removed by centrifugation at 17,000 ϫ g for 1 h. The pellet was resuspended in 20 mM potassium phosphate buffer (pH 7.5) containing 0.1 mM EDTA and 20% glycerol and the CYP4A11 solubilized by the addition of 1% Emulgen 913. The solution was stirred gently for 1 h at room temperature. The nonsolubilized protein was removed by centrifugation at 100,000 ϫ g for 1 h.
Binding of Substituted Imidazoles to CYP4A11 and L131F-Binding of substituted imidazoles to CYP4A11 and its L131F mutant was monitored by modification of a previous procedure (15). The substituted imidazoles studied were Im, 1-MeIm, 1-PhIm, 1-BzIm, 4-MeIm, 4-PhIm, 4,5-diClIm, 4,5-diBrIm, and 4,5-diPhIm. Binding of the imidazoles was monitored in 500-l samples of CYP4A11 and its L131F mutant prepared as follows. The ferric sample had no additions prior to addition of the imidazoles. The ferrous sample was prepared by addition of sodium dithionite to 10 mM of the ferric sample. Additional samples of the ferric and ferrous proteins were prepared with an NADPH regenerating system consisting of 6 units of glucose-6-phosphate dehydrogenase, 7.8 mg of glucose 6-phosphate, and 5 g of dilauroylphosphatidylcholine with either NADPH-cytochrome P450 reductase (in quantities equal to that of the P450 in the sample) or 0.1 mM NADPH also present. In addition, one sample of the ferric protein was prepared with dilauroylphosphatidylcholine, the regenerating system, the reductase, and NADPH all present. This resulted in reduction of the ferric to the ferrous protein without the addition of dithionite. The difference spectra from 350 -700 nm were monitored until no further changes were observed. Spectroscopic analysis of the CYP4A11 system under aerobic versus anaerobic conditions demonstrated that the dithionite-reduced enzyme even under aerobic conditions was completely in the ferrous state. Similar experiment showed that, even under aerobic conditions, the NADPH-reduced enzyme was largely (ϳ85%) in the ferrous state.

Expression and Spectroscopic Characterization of CYP4A11
and L131F-The mutation to be made in CYP4A11 was chosen by alignment of the CYP4A11 sequence with that of P450 BM-3 , for which two independent crystal structures are available (18,22). The sequence alignments were done with the GCG, Clustal V, and PIMA alignment programs (30,31). The fact that P450 BM-3 exhibits ϳ30% sequence similarity to the CYP4A enzymes and catalyzes fatty acid hydroxylation (20,21) suggest that it is the best available crystallographic model for CYP4A11. It has been shown that an F87A mutation in P450 BM-3 alters the regiospecificity of substrate hydroxylation. Whereas wild-type P450 BM-3 catalyzes the :-1:(-2 ϩ -3)hydroxylation of lauric acid in a Ͻ1:30:70 ratio, the F87A mutant predominantly (Ͼ90%) -hydroxylates lauric acid (23). This finding is consistent with the proposal that Phe-87 helps to sequester the terminal methyl group of the substrate and thereby controls the hydroxylation regiospecificity (23). Phe-87 lies in one of the domains predicted by Gotoh from sequence alignments of the CYP2 proteins with P450 cam to have direct contacts with the substrate (32). Furthermore, x-ray crystallography has shown that substrate binding induces a conformational change that causes Phe-87 to flip approximately 90 o from almost perpendicular to almost parallel (and in close proximity) to the heme plane, a position that interferes with substrate access to the iron atom (22). We have therefore chosen to mutate Leu-131 in CYP4A11, which corresponds to Phe-87 in P450 BM-3 , to a phenylalanine (Fig. 1).
Wild-type CYP4A11 and its L131F mutant were heterologously expressed in E. coli with a carboxyl-terminal His 6 -tail to facilitate purification of the proteins on a nickel affinity column. Expression levels for both the wild-type and L131F proteins, as determined by measurement of the P450 chromophore, were 50 -100 nmol/l. Comparison of the activity of the purified CYP4A11 thus obtained with literature data indicates that the His 6 -tail has no obvious effect on the enzymatic activity (5,6).
Wild-type and L131F CYP4A11 have comparable Soret absorption maxima in the ferric and ferrous states, as the ferric⅐Im complexes, and as the ferrous⅐CO complexes (Table I).
No cytochrome P420 was detected in the purified preparations of either protein. The absence of a significant chromophore perturbation with respect to the wild-type suggests that the heme environment in the L131F mutant is not significantly perturbed. Neither the purified wild-type nor the L131F mutant was stable to prolonged storage at Ϫ70°C, but coordination of imidazole to the heme stabilized both proteins toward storage. Both the wild-type and L131F enzymes were stable during the ϳ30-min time period required for the present experiments (e.g. see Fig. 5).
Catalytic Activities of CYP4A11 and Its L131F Mutant-CYP4A11 and its L131F mutant catalyze the hydroxylation of lauric acid but at different rates and with different regiospecificities (Table II). The L131F mutant has no detectable -hydroxylation activity but retains an -1 hydroxylation activity comparable with that of the wild-type protein. In control experiments, no fatty acid hydroxylation was observed when CYP4A11 was left out of an otherwise complete incubation mixture even if catalase was excluded and H 2 O 2 (2 mM final concentration) was added. The K m and V max values could not be determined for the L131F mutant, because the enzyme was not saturated by the accessible lauric acid concentrations (approximately 1.2 mM), but the ratio of V max /K m calculated from the initial slope of the reaction was much smaller for the L131F mutant than for CYP4A11 (Table II). Despite major changes in the hydroxylation rate and regiospecificity, the NADPH consumption rate (approximately 200 nmol min Ϫ1 nmol Ϫ1 ) was essentially the same for the wild-type and L131F mutant.
Binding of Imidazoles to CYP4A11 and L131F-To explore the structural changes in the CYP4A11 active site caused by the L131F mutation, spectroscopic binding constants (K s ) were determined for the binding of 1-, 4-, and 4,5-disubstituted imidazoles to both CYP4A11 and its L131F mutant. The K s values are spectroscopically determined K d values and are obtained by measuring the change in the amplitude of the difference spectrum as a function of the inhibitor concentration. The spectroscopic change is associated with coordination of the imidazole to the iron atom. The K s values for the binding of substituted imidazoles to the L131F mutant were generally found to be modestly larger than for their binding to wild-type CYP4A11 except for 4,5-diPhIm, which bound much more weakly to the L131F mutant than to wild-type CYP4A1 (Fig. 2). These results suggest that the L131F mutation decreases the access the imidazoles have to the iron in the active site of CYP4A11.
The effect of reducing the iron atom on the binding of imidazoles to CYP4A11 has been investigated to determine whether the active site topology is sensitive to the protein redox state (Fig. 3, Table III). Comparisons of the K s values for binding of substituted imidazoles to the ferric and the dithionite-reduced ferrous proteins demonstrate that the change in the iron oxidation state, by itself, has little or no effect on ligand binding (Fig. 3). However, when CYP4A11 is enzymatically reduced by incubation with cytochrome P450 reductase and an NADPH regenerating system, the K s values for all the imidazoles except 1-PhIm increase, in some cases dramatically.
Multiple control experiments establish that none of the components of the reconstitution mixture individually or in combination have a similar effect on the K s values (Fig. 3, Table III). It is particularly notable that the ferrous enzyme obtained with dithionite in the presence of cytochrome P450 reductase, but absence of NADPH, does not exhibit the altered binding of imidazoles that is found when NADPH is also present (Fig. 3). The impaired binding of imidazoles to CYP4A11 thus requires not only reduction of the iron to the ferrous state but the presence of both cytochrome P450 reductase and NADPH.
A similar analysis of the binding of substituted imidazoles to   the L131F mutant reveals a similar pattern of results (Table  IV). The principal difference is that 4,5-diPhIm, which bound very poorly to the enzymatically reduced wild-type enzyme, does not detectably bind to the enzymatically reduced L131F mutant.
Reaction with Phenyldiazene-To further characterize the active site of CYP4A11 and its L131F mutant we have examined their reactions with phenyldiazene, an agent that commonly reacts with P450 enzymes to give a stable -bonded phenyl-iron complex with an absorption maximum at 475-480 nm (33). Formation of the complex, and its subsequent rearrangement to give N-phenyl heme adducts, have provided useful information on the active site topologies of several P450 enzymes. However, incubation of CYP4A11 (Fig. 4) and its L131F mutant (not shown) with phenyldiazene (PhNϭNH) did not yield a detectable phenyl-iron complex but simply caused a gradual decrease in the Soret absorption intensity due to deg-radation of the prosthetic heme group.
Inactivation by 1-ABT-1-ABT is a mechanism-based cytochrome P450 inhibitor. The inactivation of P450 by 1-ABT involves the catalytic formation of benzyne, which either adds across two of the prosthetic heme nitrogen atoms to give an N,N-bridged porphyrin adduct (34,35) or covalently modifies the protein (36). Previous studies have demonstrated that 1-ABT inactivates the P450-dependent fatty acid -hydroxylation activity in rat microsomal preparations (36) and in vivo (37)(38)(39)(40). This inactivation was unexpected, because 1-ABT is a bulky aromatic compound with little resemblance to the normal CYP4A fatty acid substrates. To further examine this question, the susceptibilities of CYP4A11 and its L131F mutant to inactivation by 1-ABT were investigated. The enzymes were incubated in a reconstituted system with 10 mM 1-ABT prior to adding lauric acid and measuring the hydroxylation activity. Wild-type CYP4A11 was found to be inactivated in a time-and NADPH-dependent manner, but the L131F mutant was resistant to inactivation (Fig. 5). Inactivation of CYP4A11 by 1-ABT was shown not to involve modification of the heme chromophore by the finding that similar ferrous⅐CO complexes were obtained with aliquots of the enzyme taken before and after incubation with 10 mM 1-ABT under turnover conditions (Fig. 6).

DISCUSSION
Determination of the active site properties of CYP4A11 make possible a comparison of its active site with that of CYP4A1. Polyhistidine-labeled CYP4A11 has chromatographic and spectroscopic properties similar, but not identical to, those of CYP4A1 (Table I) (15). A detailed comparison of the substrate specificities of CYP4A11 and CYP4A1 is not yet possible, because data on a common set of fatty acids are not available, but both enzymes exhibit the highest activity for fatty acids approximately twelve carbons in length with a lower activity for both shorter and longer substrates (2,6,41). The dissociation constants for the binding of a common set of substituted imidazoles to both enzymes show that the imidazoles generally bind more tightly to CYP4A11 (Fig. 7) than to CYP4A1 (15), but similar trends are observed with both enzymes as the substituents are varied. Thus, the K s value for unsubstituted imidazole is 0.4 mM for CYP4A11 and 1.4 mM for CYP4A1, the worst ligand for both enzymes is 1-MeIm, and 4-MeIm is bound by both enzymes with roughly the same affinity as unsubstituted imidazole (Fig. 7). The most tightly bound ligands are the mono-aryl-substituted imidazoles, presumably because they represent an optimum balance of lipophilicity and steric size. The advantage of increased lipophilicity is counterbalanced by increasing steric interactions as the size of the substituent increases, resulting eventually in the more weakly bound disubstituted imidazoles (Fig. 7). 4,5-DiClIm is an abnormally poor ligand for CYP4A1, but its binding to CYP4A11 is in line with that of the other two disubstituted imidazoles. These results suggest that the active sites have topological similarities but indicate that the CYP4A11 active site is more sterically unencumbered or more lipophilic than that of CYP4A1.
Despite the inference from the imidazole spectroscopic dissociation constants that the CYP4A11 active site is somewhat more accessible than that of CYP4A1, the iron atom in both enzymes appears to be relatively inaccessible. The reaction of CYP4A11 with phenyldiazene, like that of CYP4A1 (15), results in degradation of the heme chromophore rather than the detectable formation of a phenyl-iron complex (Fig. 4). The two CYP4A enzymes are differentiated by this susceptibility to degradation by phenyldiazene from the majority of P450 enzymes, which react with this agent to give stable phenyl-iron complexes (33). The formation of a phenyl-iron complex re-

FIG. 2. Comparison of the K s values for the binding of substituted imidazoles to CYP4A11 (solid bars) and its L131F mutant (open bars).
FIG. 3. Binding of substituted imidazoles to ferric and ferrous CYP4A11 in the presence and absence of individual components of the reconstituted enzyme system. Fe 3ϩ only, ferric enzyme alone; Fe 3ϩ -NADPH, completely reconstituted system with ferric enzyme minus NADPH; Fe 3ϩ -reduct, completely reconstituted enzyme minus the P450 reductase; Fe 2ϩ only, ferrous enzyme obtained by dithionite reduction without other components of the system; Fe 2ϩ -NADPH, completely reconstituted dithionite-reduced ferrous enzyme minus NADPH; Fe 2ϩ -reduct, completely reconstituted dithionite-reduced ferrous enzyme minus P450 reductase; Fe 2ϩ all, completely reconstituted ferrous enzyme reduced by NADPH. quires sufficient space above the iron atom for the reaction to take place and for the phenyl-iron complex to be assembled. The failure to form such a complex is consistent with an active site in which formation of the phenyl-iron complex is blocked by steric constraints. The degradation of the heme chromophore observed in the CYP4A11 phenyldiazene reaction is likely to result from reaction of the heme with peroxides or other reactive oxygen species produced during decomposition of the phenyldiazene.
A major difference between CYP4A11 and CYP4A1 is the comparative sensitivity of these two enzymes to catalysis-dependent inactivation by 1-ABT. Purified, recombinant CYP4A11 reconstituted with cytochrome P450 reductase and cytochrome b 5 is inactivated in a time-and turnover-dependent manner by preincubation with 1-ABT (Fig. 5). Retention of the full heme chromophore absorbance in the inactivated protein indicates that the inactivation, contrary to what is observed with many P450 enzymes (34,35), is due to a reaction of the catalytically generated benzyne with the protein rather than the heme group. In contrast, purified CYP4A1 is insensitive to incubation with 1-ABT under the same conditions, even though its D323E mutant is readily inactivated (15). The sensitivity of CYP4A11, but not CYP4A1, to inactivation by 1-ABT indicates that the CYP4A11 active site is more accessible to substrates without a structural relationship to fatty acids, in accord with the evidence from the imidazole binding studies that the CYP4A11 site is somewhat larger or more open than the CYP4A1 site.   Important evidence with respect to the CYP4A11 active site is provided by the finding that the L131F mutation makes CYP4A11 resistant to inactivation by 1-ABT (Fig. 5). This observation is consistent with the finding that the mutation causes a decrease in the binding of most substituted imidazoles, and a major decrease in the binding of 4,5-diPhIm, to the enzyme (Fig. 2). If Leu-131 occupies the same site in CYP4A11 as Phe-87 does in P450 BM-3 (Fig. 8), mutation of the leucine to the larger phenylalanine would be expected, as found, to decrease access of heterocyclic agents to the heme prosthetic group. The results suggest that Leu-131, in fact, occupies a position in the CYP4A11 active site comparable with that of Phe-87 in P450 BM-3 .
An F87A mutation in P450 BM-3 shifts the regiospecificity of the enzyme from (-1)-, (-2)-, and (-3)-hydroxylation to -hydroxylation of fatty acids (23). Mutation of Leu-131, the CYP4A11 residue equivalent to Phe-87 in P450 BM-3 (Fig. 1), to a phenylalanine also profoundly alters the rate and regioselectivity of the human fatty acid hydroxylase (Table II). The -hydroxylation activity is completely suppressed by the mutation without a compensating increase in (-1)-hydroxylation. In fact, the enzyme retains a wild-type level of (-1)-hydroxylation activity (Table II). Because the L131F mutant oxidizes NADPH at essentially the same rate as wild-type CYP4A11, the decrease in the rate of fatty acid -hydroxylation is due to uncoupled reduction of oxygen to H 2 O 2 and/or H 2 O (Table II).
The uncoupled reduction of oxygen by P450 enzymes is well established even though the mechanisms that control partitioning between substrate hydroxylation and uncoupled oxygen reduction remain unclear. The available evidence suggests that uncoupling increases when proton delivery to the iron-bound dioxygen molecule is impaired or is not well controlled (42). The L131F-mediated increase in the uncoupling of CYP4A11 suggests that the larger phenylalanine side chain perturbs the hydrogen bonding network that is responsible for proton delivery during catalytic turnover. It may do this by directly altering the orientation of the substrate or catalytic residues or by causing such changes through an allosteric mechanism. The surprise is that the uncoupling reaction is enhanced at the expense of thebut not (-1)-hydroxylation of lauric acid. If the same catalytic intermediate is involved in both reactions, uncoupling might be expected to affect both reactions to the same extent. However, it is conceivable that (-1)-hydroxyla- tion is less affected, because thermodynamically it is an easier reaction than -hydroxylation and therefore competes more effectively with uncoupled reduction of the activated ferryl species to water (42). Alternatively, the substrate may be bound in two different orientations forversus (-1)-hydroxylation, possibly with an accompanying conformational alteration in the active site, and only one of these binding modes leads to enhanced uncoupling in the presence of the L131F mutation. In either case, the results provide strong evidence that Leu-131 is located in the active site in a position similar to that occupied by Phe-87 in P450 BM-3 and that P450 BM-3 is a useful template on which to model the CYP4A11 active site.
The binding of substituted imidazoles to CYP4A11 and its L131F mutant is influenced by both the redox state of the protein and the presence or absence of cytochrome P450 reductase and NADPH (Fig. 3). Minor differences are seen in the K s values for the binding of Im, 1-MeIm, 4-MeIm, and 4,5-diPhIm to (a) the ferric protein alone or in the presence of the other components of the normal incubation mixture except NADPH or (b) the dithionite-reduced ferrous protein alone or in the presence of the other incubation components except NADPH. However, the K s values are much higher for the binding of the same imidazoles to the ferrous enzyme when both P450 reductase and NADPH are present (Fig. 3). The difference in binding is most marked for 4,5-diPhIm, the bulkiest and most lipophilic of the ligands. Although a similar difference in K s values is not observed for 1-PhIm, 4-PhIm, and 1-BzIm, the most tightly bound ligands, the results with the other six ligands suggest that the protein undergoes a conformational change when it is reduced but only if the enzyme is complexed with P450 reductase and NADPH. The influence of NADPH in this process could reflect a direct allosteric effect or could result from structural alterations associated with reduction of the flavin groups in the reductase. The results provide the first evidence for a redox-dependent conformational change in a mammalian P450 enzyme. The results with CYP4A11 bring to mind the crystallographic demonstration that a major conformational change takes place when a fatty acid is bound to P450 BM-3 and the NMR evidence that a second conformational change occurs when the iron atom in the substrate-bound enzyme is reduced (22,24). The conformational change(s) observed in CYP4A11 may or may not resemble those observed with P450 BM-3 , but their existence provides a further structural link between the two proteins.
In sum, the present studies demonstrate that the CYP4A11 active site is more accessible to exogenous ligands and inhibitors than the CYP4A1 site, establish that the residue at position 131 plays a key role in determining both the degree of coupling and the regiochemistry of fatty acid hydroxylation, show that the enzyme undergoes a redox-dependent conformational change, and confirm the use of P450 BM-3 as a structural template for CYP4A11.