Crystal Structure of H2O2-dependent Cytochrome P450SPα with Its Bound Fatty Acid Substrate

Cytochrome P450SPα (CYP152B1) isolated from Sphingomonas paucimobilis is the first P450 to be classified as a H2O2-dependent P450. P450SPα hydroxylates fatty acids with high α-regioselectivity. Herein we report the crystal structure of P450SPα with palmitic acid as a substrate at a resolution of 1.65 Å. The structure revealed that the Cα of the bound palmitic acid in one of the alternative conformations is 4.5 Å from the heme iron. This conformation explains the highly selective α-hydroxylation of fatty acid observed in P450SPα. Mutations at the active site and the F–G loop of P450SPα did not impair its regioselectivity. The crystal structures of mutants (L78F and F288G) revealed that the location of the bound palmitic acid was essentially the same as that in the WT, although amino acids at the active site were replaced with the corresponding amino acids of cytochrome P450BSβ (CYP152A1), which shows β-regioselectivity. This implies that the high regioselectivity of P450SPα is caused by the orientation of the hydrophobic channel, which is more perpendicular to the heme plane than that of P450BSβ.

olism of many exogenous and endogenous compounds (1)(2)(3)(4). X-ray crystal structure analysis is one of the most powerful methods for visualizing the structures of P450s and their interactions with substrates in the heme cavity at the atomic level. Since the first crystal structure of P450, P450 cam (CYP101A1) was reported by Poulos et al. (5), the crystal structures of P450s from mammals (6 -14), archaea (15)(16)(17), and bacteria (18 -27) have been reported, and interactions between their substrates and amino acid residues at substrate recognition sites have been clarified. Most P450s accomplish monooxygenation by reductive activation of molecular oxygen using NADPH or NADH to produce compound I (oxoferryl porphyrin cation radical). P450s also use H 2 O 2 to generate compound I, but the efficiency of this reaction is poor compared with that of reductive activation of molecular oxygen. In 1994, Matsunaga et al. (28) isolated P450 SP␣ (CYP152B1) from Sphingomonas paucimobilis and reported that it exclusively uses H 2 O 2 as the oxidant and catalyzes ␣-selective (100%) hydroxylation of long alkyl chain fatty acids (29). Although P450 SP␣ is the first P450 to be classified as a family of H 2 O 2 -dependent P450, its crystal structure has not been determined despite its potential as a biocatalyst. The first crystal structure of H 2 O 2 -dependent P450, P450 BS␤ (CYP152A1), which has 44% amino acid identity to P450 SP␣ , was reported in 2003 by Lee et al. (30). The crystal structure of a substrate-bound form of P450 BS␤ (Protein Data Bank code 1IZO) revealed that P450 BS␤ lacks general acid-base residues around the distal side of the heme, although this arrangement is highly conserved among peroxidases and peroxygenases (31)(32)(33)(34)(35). Instead of the general acid-base residues, the terminal carboxylate group of the bound fatty acid interacts with the guanidinium group of Arg 242 located near the heme group. The distance between an oxygen atom of the carboxylate group of palmitic acid and the heme iron is 5.3 Å, which is similar to that observed in chloroperoxidase (CPO) from Caldariomyces fumago; the distance between an oxygen atom of glutamic acid side chain and the heme iron is 5.1 Å (34,35). The location of the carboxylate group of palmitic acid bound to P450 BS␤ suggests that the general acid-base function for the facile formation of compound I is accomplished by the carboxylate group of the substrate (Scheme 1). Recently, we have shown that P450 BS␤ is able to catalyze H 2 O 2 -dependent monooxygenation of foreign compounds such as styrene and ethylbenzene in the presence of a carboxylic acid with a short alkyl chain (C 4 -C 10 ), a so-called "decoy molecule" (36). The crystal structure of a heptanoic acid (C 7 )-bound form of P450 BS␤ was analyzed, and an interaction between Arg 242 and the carboxylate group of heptanoic acid was detected. We also resolved the crystal structure of the substrate-free form of P450 BS␤ and found that binding of fatty acid or substrate analogues did not induce any notable structural change, whereas the substrate-free form of P450 BS␤ never reacts with H 2 O 2 (37). These observations further confirm that substrate binding initiates the formation of compound I via a salt bridge between Arg 242 and the carboxylate group at the active site.
P450 BS␤ oxidizes the ␣and ␤-positions of fatty acids in a 40:60 ratio, whereas P450 SP␣ exclusively oxidizes the ␣ position. To elucidate the cause of this selectivity, we need to study the crystal structures of both enzymes. Although the crystal structure of the substrate-bound form of P450 BS␤ is known, that of P450 SP␣ has not been resolved. Therefore, we crystallized P450 SP␣ and succeeded in preparing high quality crystals of P450 SP␣ . Herein we describe the x-ray crystal structure of P450 SP␣ containing a fatty acid at a resolution of 1.65 Å and examine enzymatic properties of its mutants to study its highly selective ␣-hydroxylation of fatty acids. We also compare the structure of P450 SP␣ with that of P450 BS␤ in the context of similarities and differences among H 2 O 2dependent P450s.

EXPERIMENTAL PROCEDURES
Crystallization of P450 SP␣ -P450 SP␣ WT was concentrated to 13.4 mg/ml in 50 mM MES (pH 7.0) containing 20% (v/v) glycerol by centrifugation using Amicon Ultra filter units (Millipore, Co.). A 2-l aliquot of the concentrated P450 SP␣ solution was mixed with 2 l of a reservoir solution composed of 0.1 M HEPES (pH 7.0) and 35% (v/v) MPD. Crystals of P450 SP␣ were grown by a sitting-drop vapor diffusion method at 20°C for 6 days. The P450 SP␣ L78F and F288G mutants were crystallized under the same conditions used for the WT.
Data Collection, Phasing, and Refinement of P450 SP␣ -Crystals were flash-cooled in liquid nitrogen. X-ray diffraction data sets were collected on a beam line BL41XU instrument equipped with an ADSC Quantum 315 CCD detector at the RIKEN SPring-8 (Hyogo, Japan) with a 1.0 Å wavelength at 100 K. The HKL2000 (38) program was used for integration of diffraction intensities and scaling. Initial phases were calculated and refined using the SHELXE program (39) and the hkl2map graphical interface (40). In the calculated electron density, the main chain was clearly traceable, and the initial polypeptide chain was built using ARP/wARP (41). Model building and refinement were performed using COOT (42), CNS (43), and REFMAC5 (44). TLS refinement (45) was performed in the final stages of the refinement, defining each chain in the asymmetric unit as a separate TLS group. The resulting model had a final R fact of 15.1% and an R free of 17.3% (Table 1). The final model consisted of one polypeptide chain with residues 9 -415 of P450 SP␣ , one heme, one palmitic acid, one MPD, and 371 water molecules. Structure validation was performed using SCHEME 1. A proposed catalytic reaction mechanism for hydroxylation of long alkyl chain fatty acid. For the generation of compound I (oxoferryl porphyrin -cation radical), the carboxylate group of the fatty acid (blue) that serves as a general acid-base catalyst first accepts a proton from H 2 O 2 , producing the ferric hydroperoxy complex (Fe ϩ3 -OOH). Subsequently, a proton is donated to the distal oxygen atom of the ferric hydroperoxy complex followed by the O-O bond cleavage to produce compound I.
WHAT-IF (46) and PROCHECK (47). X-ray diffraction data sets of the L78F and F288G mutants were collected using a beam line BL26B1 instrument equipped with an ADSC Quantum 210 CCD detector at SPring-8. The structures of L78F and F288G were solved using a molecular replacement method using MOLREP (48), followed by refinement using COOT and REMAC5. The final refinement statistics are summarized in Table 1.
Structural Analysis of P450 SP␣ and P450 BS␤ -Electrostatic potentials were calculated using GRASP2 (49). Probe-occupied voids were calculated using VOIDOO (50), and a probe of 1.1 Å and a grid mesh of 0.3 Å were used unless otherwise specified. The accessible channels were calculated using CAVER (51). All of the protein figures were depicted using PyMOL (52).
Hydroxylation of Myristic Acid-The standard reaction mixture contained 0.1 M potassium phosphate (pH 7.0), 0 -120 M myristic acid (C 14 ) (0 -60 M for F288G and A172F/F288G), 50 nM P450 SP␣ or P450 BS␤ , and 200 M H 2 O 2 in a total volume of 1 ml. The reaction mixture was incubated at 37°C for 1 min, and then the reaction was quenched by adding 500 l of dichloromethane followed by vigorous mixing. After the addition of 12-hydroxydodecanoic acid as an internal standard, the products were extracted with dichloromethane. For derivatization of the extract, 50 l of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) containing 1% (v/v) trimethylchlorosilane (TMCS) was added, and the mixture was incubated in the dark at room temperature for 2 h. The derivatized products were analyzed using a Shimadzu GC-17A (Shimadzu Corp., Kyoto, Japan) equipped with a Shimadzu GC/MS-QP5000 and Rxi TM -5ms capillary column (30 m ϫ 0.25 mm; Restek Corp., Bellefonte, PA) to identify the products. The GC/MS analytical conditions were as follows: column temperature, 50°C (1 min) to 40°C/min (5 min) to 250°C (8 min); injection temperature, 250°C; interface temperature, 280°C; carrier gas, helium; flow rate, 0.9 ml/min, mode, split mode; and split ratio, 1/50. To quantify the products, derivatization of the extract was performed by adding 9-anthryldiazomethane and incubating the solution in the dark at room temperature for 1 h. For quantification of the products, reverse phase HPLC analysis was performed using an Inertsil ODS-3 column (4.6 mm ϫ 250 mm; GL Sciences, Inc., Tokyo, Japan) installed on a Shimadzu SCL-10A VP system controller equipped with Shimadzu LC-10AD VP pump systems, a Shimadzu RF-10A XL fluorescence spectrometer, a Shimadzu CTO-10A VP column oven, and a Shimadzu DGU-12A degasser. The HPLC analytical conditions were as follows: flow rate, 1.0 ml/min; acetonitrile/water ϭ 99/1; column temperature, 30°C; excitation wavelength, 365 nm, emission wavelength, 412 nm; and retention times, 12-hydroxydodecanoic acid (6.41 min), ␤-OH C 14 (10.7 min), ␣-OH C 14 (11.9 min), and C 14 (21.5 min). Chiral separation of the products was performed on a CHIRALPAK AD-RH column (Daicel Chemical Industries, Ltd., Osaka, Japan) installed on the same reverse phase HPLC system as in the case of the quantification. The absolute configuration was assigned by comparison of the product ratios in the hydroxylation of C 14 by P450 SP␣ WT (53) and P450 BS␤ WT (54). The HPLC conditions for the chiral separation were as follows: flow rate, 0.9 ml/min; linear gradient, MeOH/water ϭ 85/15 (0 -10 min) to 100/0 (100 -120 min); column temperature, 40°C; excitation, 365 nm; emission, 412 nm; retention times, (R)-␣-OH C 14 (34. UV-visible and EPR Measurements-UV-visible spectra were recorded using a Shimadzu UV-2400 PC spectrophotometer at room temperature. X-band EPR spectra were recorded using an E500 X-band CW-EPR instrument (Bruker, Ettlingen, Germany) at 10 K. A cryostat (ITC503; Oxford Instruments Co., Abingdon, UK) was used for measurements at low temperatures.
where F o and F c are the observed and calculated structure factor amplitudes, respectively. d R free was calculated as the R fact for 5% of the reflections that were not included in the refinement. e RMSD, root mean square deviation.

RESULTS AND DISCUSSION
Overall Structure and Substrate Binding-The structure of P450 SP␣ was resolved at a resolution of 1.65 Å (Fig. 1). One molecule was observed in the asymmetric unit. The overall structure exhibited typical P450 folding with 17 ␣ helices and three ␤ sheets. It has a trigonal prism-shaped structure with the heme buried deep inside the protein. The I helix lays across the interior of the P450 molecule on the distal side of the heme group. Two channels connecting the active site cavity with the protein surface were identified (Fig. 1). Channel I is composed of hydrophobic residues (Ile 73 , Leu 77 , Leu 78 , Phe 169 , Ala 172 , Ala 245 , Phe 287 , Phe 288 , Pro 289 , Leu 398 , and Pro 399 ) ( Fig. 2A). Channel II includes hydrophilic residues (Gln 84 and Asn 238 ) as its constituent residues. A cluster of water molecules with a hydrogen-bonding network was observed in Channel II (Fig.  2D). We expect that Channel II would be used for the ingress of H 2 O 2 and the egress of water during the reaction. Phe 288 is located at the border of the two channels, but the two channels are not clearly separated because the entrances of the channels are wide (Figs. 1, B and C, and 3A).
Although no substrates were added to the purified P450 SP␣ , the initial 2F o Ϫ F c electron density map showed a long contin-uous electron density in Channel I ( Fig. 2A). One of the ends of this electron density, located near Arg 241 in the active site, has a Y-shape. Because the shape of this electron density is very similar to that of a long alkyl chain fatty acid observed in P450BM3 (21) and P450 BS␤ (30), we assumed that this electron density corresponds to a long alkyl chain fatty acid originating from Escherichia coli cells (55). Indeed, GC/MS analysis of the extract of the purified P450 SP␣ with dichloromethane showed that palmitic acid and stearic acid were coexistent, even after purification (supplemental Fig. S1). It was difficult to deduce the length of the alkyl chain of the fatty acid based on the electron density of the substrate(s) because the electron density of the substrate was shorter than the alkyl chain of palmitic acid, possibly because of disordering. Therefore, we tentatively assigned this electron density to palmitic acid. In addition, because the Y-shaped electron density adjacent to the Arg 241 was accompanied by an additional electron density, two alternative conformations with occupancies of 0.7 (Conformation A) and 0.3 (Conformation B) were placed and refined (Fig. 2, B and C). The terminal alkyl chain of palmitic acid in the final structure is highly disordered, indicating that the terminal alkyl chain is loosely fixed. It is noteworthy that the AЈ-helix, BЈ-he- lix, and F-G loop have relatively high B-factors (Fig. 3B) and that the entrance of Channel I is open wide (Fig. 3A), suggesting that this region is flexible even though the substrate was accommodated. The substrate access channel and B-factor of P450 BS␤ are shown in supplemental Fig. S2 for comparison.
The charge distribution on the surfaces of the proximal and distal sides of P450 SP␣ is shown in Fig. 4. In contrast to the charge distribution typical of P450s such as P450BM3 (56, 57), negative surface charges were observed on the proximal side of P450 SP␣ . A positive surface potential on the proximal side of regular P450 is important for the recognition of reductases in the electron transfer step of oxygen activation in the P450 catalytic cycle (58). The negative surface potential on the proximal side of P450 SP␣ may preclude binding of a reductase. This unique charge potential distribution of P450 SP␣ is an indication that P450 SP␣ does not need binding of a reductase and prefers  the H 2 O 2 shunt pathway. P450 BS␤ also has no positively charged region on the surface of the proximal side. In addition, a long loop structure was observed on the proximal side ( 349 QGGGDHYLGHRC 361 ) (Fig. 5), whereas P450s that require electron transfer from reductases have short loop structures. P450 cam , for example, has a short loop structure, 350 FGHGSHLC 357 . P450 BS␤ ( 352 QGGGHAEKGHRC 363 ) (30) and allene oxide synthase ( 455 WSNGPETETPTVGNKQC 472 ) (59) also have long loop structures at the proximal side of the heme, implying that the long loop structure is a common feature among P450s that do not require electron transfer systems for the activation of molecular oxygen.
Active Site Structure-At the active site, Arg 241 is located above the heme, and its guanidinium group interacts with the carboxylate group of palmitic acid ( Fig. 2A); the distances between two oxygen atoms of the carboxylate group and the guanidinium group of Arg 241 (N 2 and N ⑀ ) are 2.9 and 3.0 Å in Conformation A (Fig. 2B) and 3.3 and 3.2 Å in Conformation B (Fig. 2C), respectively. In contrast to other heme enzymes that utilize H 2 O 2 , P450 SP␣ lacks general acid-base residues around the distal side of the heme. As an alternative, the terminal carboxylate group of palmitic acid is located above the heme. The distance between the oxygen atoms close to the heme iron is 5.2 Å for Conformation A and 5.5 Å for Conformation B, indicating that the location of the oxygen atom is similar to that of the glutamic acid moiety of CPO (34,35) and that of the terminal carboxylate group of palmitic acid observed in P450 BS␤ . These observations indicate that participation of the terminal carboxylate group of the fatty acid in the generation of active species using H 2 O 2 (Scheme 1) is common among H 2 O 2 -dependent P450s. The distal side of the heme is hydrophilic because of Gln 84 , Asp 238 , Arg 241 , and the carboxylate group of palmitic acid (Fig. 2D). The polar environment is expected to facilitate the heterolytic cleavage of the O-O bond of H 2 O 2 to generate compound I, as is proposed for heme peroxidases such as cytochrome c peroxidase (31,32), HRP (33), CPO (34,35), and myoglobin mutants (60,61). A water molecule is located 2.1 Å away from the heme iron and could function as a sixth ligand on the heme even though the palmitic acid occupies the distal side of the heme cavity ( Fig. 2A). The UV-visible spectra of P450 SP␣ in the absence and presence of 120 M of myristic acid showed a Soret absorption peak at 417 nm, which is consistent with a typical six-coordinate low-spin ferric heme (Fig. 6). The EPR spectrum of the substrate-free form of P450 SP␣ showed a ferric low spin state having g value 2.59 (g z ), 2.25 (g y ), and 1.85 (g x ) (Fig. 7), suggesting that the electronic environment of the heme iron of P450 SP␣ resembles that of CPO (2.61 (g z ), 2.26 (g y ), 1.83 FIGURE 4. Electrostatic potential surface of P450 SP␣ . Electrostatic surfaces of P450 SP␣ were calculated using GRASP2 (49). The negatively charged surface is represented in red, and the positively charged surface is represented in blue. Palmitic acid is shown as a yellow stick model.   (g x ), pH 5.2) (62) rather than those of bacterial P450s such as P450 cam (2.45 (g z ), 2.26 (g y ), 1.91 (g x )) (63) and P450BM3 (2.42 (g z ), 2.26 (g y ), 1.92 (g x )) (64). The EPR spectral change of P450 SP␣ upon addition of myristic acid (120 M) was very small, indicating that the low spin state is essentially retained irrespective of substrate binding. Minor signals at g ϭ 2.67 in the substrate free-form and at g ϭ 2.53 in the myristic acid-bound form might reflect P420 species of P450 SP␣ (supplemental Fig. S5), whereas signals are different from that of P420 species of P450 cam (2.46 (g z )) (64).
Regioselectivity for Hydroxylation of Fatty Acid-P450 SP␣ exclusively catalyzes the hydroxylation of fatty acid at the C ␣ position and produces the corresponding ␣-hydroxy fatty acid, whereas P450 BS␤ produces ␣ and ␤ hydroxy products in the ratio of 43:57. In the crystal structure of P450 SP␣ , the distances of the C ␣ carbon and the C ␤ carbon in Conformation B from the heme iron are 4.5 and 5.5 Å, respectively. Because the C ␣ carbon in Conformation B is clearly close to the iron atom, and the distance of 4.5 Å agrees well with the distance between the C5 position of d-camphor and the heme iron in P450 cam (65), Conformation B is expected to produce the ␣-hydroxy fatty acid selectively. Because the C ␣ and C ␤ carbons in Conformation A are both far away from the heme iron in respect of the hydroxylation reaction, we assume that Conformation A is a nonproductive conformation. In the crystal structure of P450 BS␤ , the C ␣ and C ␤ carbons of palmitic acid are located at distances of 5.0 and 6.2 Å from the heme iron, respectively. The substrate observed in P450 BS␤ needs to be closer to the heme iron to be hydroxylated, as was observed for P450BM3 (66). The C ␣ and C ␤ carbons of the possible productive conformation of palmitic acid in P450 BS␤ may be equally close to the heme iron.
Structural Comparison of P450 SP␣ with P450 BS␤ -To gain further insight into H 2 O 2 -dependent P450s, the structure of P450 SP␣ was compared with that of P450 BS␤ . The structure of P450 SP␣ is superimposed on that of P450 BS␤ in Fig. 8. Except for the BЈ helix and the F-G loop, the overall structures are well superposed with a root mean square deviation value of 1.4 Å. The locations of Channel I are notably different, resulting in different locations for the bound palmitic acid. The palmitic acid is more perpendicular to the heme plane in P450 SP␣ than in P450 BS␤ . The active site cavity of P450 SP␣ is smaller than that of P450 BS␤ (supplemental Fig. S3). The smaller active site cavity is mainly due to the presence of Phe 288 in the heme cavity of P450 SP␣ . The corresponding amino acid residue in P450 BS␤ is Gly 290 . The effect of Phe 288 and the location of Channel I are discussed in the next section.
Hydroxylation of Myristic Acid by Mutants-We carried out mutagenesis study to elucidate the structural requirement for the ␣-selective reaction of P450 SP␣ . Based on the comparison between amino acid residues in the active sites of P450 SP␣ and P450 BS␤ , two amino acid residues in the active site of P450 SP␣ , Phe 288 and Leu 78 , were mutated to the corresponding P450 BS␤ residues and vice versa. The active site cavity of P450 SP␣ is smaller than that of P450 BS␤ , mainly because of the side chain of Phe 288 (supplemental Fig. S3), which interacts directly with the fatty acid. Although Leu 78 does not interact with the fatty acid, it is located at the border of the channels and may affect the catalytic reaction. Three mutants of P450 SP␣ , L78F, F288G, and L78F/F288G, and three P450 BS␤ mutants, F79L, G290F, and F79L/G290F, were prepared and their catalytic activities, regioselectivities, and stereoselectivities were examined (Table 2). P450 BS␤ mutants F79L and G290F had 75 and 95% ␣ selectivity, respectively, indicating that the regioselectivity of P450 BS␤ was greatly altered. The substitution of Gly 290 with Phe must induce a conformational change in myristic acid, and the C ␣ carbon is expected to move closer than the C ␤ carbon to the heme iron. In sharp contrast with the P450 BS␤ mutants, no clear differences in regioselectivity were observed for P450 SP␣ mutants, whereas the amino acids at the active site were replaced with the corresponding amino acids of P450 BS␤ , which has ␤-regioselectivity. Three mutants of P450 SP␣ showed Ͼ99% ␣ selectivity. To further investigate the regioselectivity of P450 SP␣ , two mutants of P450 SP␣ , A172F and A172F/F288G, were prepared. Ala 172 located in the F-G loop seems to be important for controlling fatty acid conformation. However, the regioselectivity was not affected by these mutagenesis experiments, indicating that the mutagenesis at the active site and at the F-G loop does not alter the position of bound palmitic acid. The x-ray crystal structures of the L78F and F288G mutants revealed that there is little difference in the location of the bound palmitic acid in Conformation B (the productive conformation) and the location of the hydrophilic channel (Fig. 9), whereas the position of the amino acids located near the active site, Met 69 , Pro 70 , and Pro 289 , are shifted ( Fig. 10 and supplemental Fig. S4). The C ␣ positions of palmitic acid in both mutants are essentially the same as in the WT. Whereas the stereoselectivity of ␣ hydroxylation of myristic acid was slightly reduced by mutagenesis (Table 2), the regioselectivity of P450 SP␣ was not affected, suggesting that the small perturbation induced by the mutagenesis is insufficient to alter the high regioselectivity of P450 SP␣ . We presume that the regioselectivity of P450 SP␣ is highly controlled by its hydropho-bic channel. The orientation of the channel (almost perpendicular to the heme plane) and the wide open entrance appear to be crucial for the high ␣-selectivity of hydroxylation. The hydrophobic channel may control the direction of the fatty acid access. Further mutagenesis studies, especially at the F and G helices and at the F-G loop region, are necessary for elucidating the mechanistic details of the highly selective ␣-hydroxylation of fatty acid catalyzed by P450 SP␣ .
Conclusion-We have determined the x-ray crystal structure of H 2 O 2 -dependent P450 SP␣ as a palmitic acid-bound form at a resolution of 1.65 Å. The crystal structure revealed that the carboxylate group of the fatty acid interacts with Arg 241 , which is located above the heme. Previous studies on the reaction mechanism of P450 BS␤ suggested that the carboxylate group of the fatty acid serves as a general acid-base catalyst for the generation of compound I using H 2 O 2 . Our crystal structure study confirms that this substrate-assisted activation mechanism is also conserved in P450 SP␣ , indicating that the substrate-assisted activation mechanism is common in the H 2 O 2 -dependent P450-catalyzed hydroxylation reaction with fatty acids. Notably, a water molecule was observed as the sixth ligand of the heme iron, even in the presence of palmitic acid. Consistent   with the crystal structure, the ferric low spin state of P450 SP␣ was retained irrespective of the substrate binding. These results also indicate that the shift in redox potential of the heme that is induced by substrate binding, which is generally indispensable for the reductive activation of molecular oxygen, is not essential for the H 2 O 2 -dependent P450s. Crystallographic studies on substrate binding revealed that the C ␣ carbon of the bound palmitic acid in Conformation B is situated close to the heme iron (4.5 Å). This conformation explains the highly selective ␣-hydroxylation of fatty acid. Surprisingly, mutations at the active site and at the F-G loop of P450 SP␣ did not impair the high regioselectivity. The crystal structures of the L78F and F288G mutants revealed that the location of the bound palmitic acid was not affected by these mutations. These results imply that the orientation of the hydrophobic channel of P450 SP␣ , which is more perpendicular to the heme plane than that of P450 BS␤ , is crucial for the highly selective ␣-hydroxylation.
Although further mutagenesis studies are required to fully understand the high regioselectivity of P450 SP␣ , the structural studies reported here contribute to a better understanding of the relationship between the structure and function of H 2 O 2dependent P450s at the atomic level.