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NMR Study on the Structural Changes of Cytochrome P450cam upon the Complex Formation with Putidaredoxin

2003; Elsevier BV; Volume: 278; Issue: 41 Linguagem: Inglês

10.1074/jbc.m304265200

ISSN

1083-351X

Autores

Takehiko Tosha, Shiro Yoshioka, Satoshi Takahashi, Koichiro Ishimori, Hideo Shimada, Isao Morishima,

Tópico(s)

Mass Spectrometry Techniques and Applications

Resumo

We investigated putidaredoxin-induced structural changes in carbonmonoxy P450cam by using NMR spectroscopy. The resonance from the β-proton of the axial cysteine was upfield shifted by 0.12 ppm upon the putidaredoxin binding, indicating that the axial cysteine approaches to the heme-iron by about 0.1 Å. The approach of the axial cysteine to the heme-iron would enhance the electronic donation from the axial thiolate to the heme-iron, resulting in the enhanced heterolysis of the dioxygen bond. In addition to the structural perturbation on the axial ligand, the structural changes in the substrate and ligand binding site were observed. The resonances from the 5-exo- and 9-methyl-protons of d-camphor, which were newly identified in this study, were upfield shifted by 1.28 and 0.20 ppm, respectively, implying that d-camphor moves to the heme-iron by 0.15–0.7 Å. Based on the radical rebound mechanism, the approach of d-camphor to the heme-iron could promote the oxygen transfer reaction. On the other hand, the downfield shift of the resonance from the γ-methyl group of Thr-252 reflects the movement of the side chain away from the heme-iron by ∼0.25 Å. Because Thr-252 regulates the heterolysis of the dioxygen bond, the positional rearrangement of Thr-252 might assist the scission of the dioxygen bond. We, therefore, conclude that putidaredoxin induces the specific heme environmental changes of P450cam, which would facilitate the oxygen activation and the oxygen transfer reaction. We investigated putidaredoxin-induced structural changes in carbonmonoxy P450cam by using NMR spectroscopy. The resonance from the β-proton of the axial cysteine was upfield shifted by 0.12 ppm upon the putidaredoxin binding, indicating that the axial cysteine approaches to the heme-iron by about 0.1 Å. The approach of the axial cysteine to the heme-iron would enhance the electronic donation from the axial thiolate to the heme-iron, resulting in the enhanced heterolysis of the dioxygen bond. In addition to the structural perturbation on the axial ligand, the structural changes in the substrate and ligand binding site were observed. The resonances from the 5-exo- and 9-methyl-protons of d-camphor, which were newly identified in this study, were upfield shifted by 1.28 and 0.20 ppm, respectively, implying that d-camphor moves to the heme-iron by 0.15–0.7 Å. Based on the radical rebound mechanism, the approach of d-camphor to the heme-iron could promote the oxygen transfer reaction. On the other hand, the downfield shift of the resonance from the γ-methyl group of Thr-252 reflects the movement of the side chain away from the heme-iron by ∼0.25 Å. Because Thr-252 regulates the heterolysis of the dioxygen bond, the positional rearrangement of Thr-252 might assist the scission of the dioxygen bond. We, therefore, conclude that putidaredoxin induces the specific heme environmental changes of P450cam, which would facilitate the oxygen activation and the oxygen transfer reaction. Cytochrome P450 (P450) 1The abbreviations used are: P450, cytochrome P450; ET, electron transfer; P450cam, cytochrome P450 camphor; Pdx, putidaredoxin; cyt b 5, cytochrome b 5; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect.1The abbreviations used are: P450, cytochrome P450; ET, electron transfer; P450cam, cytochrome P450 camphor; Pdx, putidaredoxin; cyt b 5, cytochrome b 5; NOESY, nuclear Overhauser effect spectroscopy; TOCSY, total correlation spectroscopy; NOE, nuclear Overhauser effect. is a heme-containing monooxygenase, which catalyzes the hydroxylation reactions of a wide variety of natural and unnatural substrates such as steroids, fatty acids, hydrocarbons, and xenobiotics (1Ortiz de Montellano P.R. Cytochrome P450 Structure, Mechanism, and Biochemistry. Plenum Press, New York1995Crossref Google Scholar). The P450 catalysis reaction requires two electrons from NADH or NADPH through the redox-linked proteins. In microsomal P450s, the electron transfer (ET) from NADPH to P450s is mediated by NADPH-P450 reductase containing FMN and FAD as the redox centers. In contrast, the electron from NADH or NADPH is sequentially transferred to ferredoxin reductase, ferredoxin, and finally P450 in mitochondrial and bacterial systems. The ET reaction between P450 and its redox partner is essential for the subsequent activation of molecular oxygen and the monooxygenation reaction.To understand the mechanism of the ET reaction in P450 and its redox partner system, the ET reaction between cytochrome P450cam (P450cam) from Pseudomonas putida, one of the bacterial P450s, and its redox partner, putidaredoxin (Pdx), has intensively been investigated. P450cam catalyzes the regio- and stereo-specific hydroxylation of its substrate, d-camphor (1Ortiz de Montellano P.R. 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Chem. 1997; 272: 21720-21725Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 14Aoki M. Ishimori K. Morishima I. Biochim. Biophys. Acta. 1998; 1386: 157-167Crossref PubMed Scopus (40) Google Scholar), and theoretical (15Pochapsky T.C. Lyons T.A. Kazanis S. Arakaki T. Ratnaswamy G. Biochimie (Paris). 1996; 78: 723-733Crossref PubMed Scopus (104) Google Scholar, 16Roitberg A.E. Holden M.J. Mayhew M.P. Kurnikov I.V. Beratan D.N. Vilker V.L. J. Am. Chem. Soc. 1998; 120: 8927-8932Crossref Scopus (74) Google Scholar) studies to clarify the molecular mechanism for the ET reaction in the P450cam·Pdx system. These studies demonstrated that Pdx interacts with the proximal surface of P450cam through the electrostatic interaction (12Unno M. Shimada H. Toba Y. Makino R. Ishimura Y. J. Biol. Chem. 1996; 271: 17869-17874Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 15Pochapsky T.C. Lyons T.A. Kazanis S. Arakaki T. Ratnaswamy G. Biochimie (Paris). 1996; 78: 723-733Crossref PubMed Scopus (104) Google Scholar, 17Furukawa Y. Ishimori K. Morishima I. Biochemistry. 2000; 39: 10996-11004Crossref PubMed Scopus (25) Google Scholar) and suggested that Arg-112 at the putative Pdx binding site forms the ET pathway in the P450cam·Pdx complex (12Unno M. Shimada H. Toba Y. Makino R. Ishimura Y. J. Biol. Chem. 1996; 271: 17869-17874Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 16Roitberg A.E. Holden M.J. Mayhew M.P. Kurnikov I.V. Beratan D.N. Vilker V.L. J. Am. Chem. Soc. 1998; 120: 8927-8932Crossref Scopus (74) Google Scholar).It was also revealed that Pdx acts as the specific electron donor for the turnover reaction of P450cam (18Lipscomb J.D. Sligar S.G. Namtvedt M.J. Gunsalus I.C. J. Biol. Chem. 1976; 251: 1116-1124Abstract Full Text PDF PubMed Google Scholar). Lipscomb et al. (18Lipscomb J.D. Sligar S.G. Namtvedt M.J. Gunsalus I.C. J. Biol. Chem. 1976; 251: 1116-1124Abstract Full Text PDF PubMed Google Scholar) carried out the mixing of oxy-P450cam with several electron donors, including the non-physiological electron donors as well as the physiological redox partner, Pdx, and examined the formation of the hydroxylation product. Low potential iron-sulfur protein such as spinach ferredoxin and bovine adrenodoxin can donate an electron to ferric P450cam (the first ET) but yield no hydroxylation products. On the other hand, the addition of rat liver cytochrome b 5 (cyt b 5) and bacterial rubredoxins to oxy-P450cam can yield the hydroxylation product, whereas these non-physiological electron donors cannot transfer the first electron to ferric P450cam. Thus, the specific complex formation between P450cam and Pdx is required for the turnover reaction.We have studied the mechanism of the specific complex formation between P450cam and Pdx (19Aoki M. Ishimori K. Fukada H. Takahashi K. Morishima I. Biochim. Biophys. Acta. 1998; 1384: 180-188Crossref PubMed Scopus (39) Google Scholar, 20Furukawa Y. Morishima I. J. Biol. Chem. 2001; 276: 12983-12990Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) and clarified that the P450cam·Pdx system exhibits unique features compared with other protein·protein ET complexes. Aoki et al. (19Aoki M. Ishimori K. Fukada H. Takahashi K. Morishima I. Biochim. Biophys. Acta. 1998; 1384: 180-188Crossref PubMed Scopus (39) Google Scholar) have reported the negative values for the changes in the enthalpy and entropy for the formation of the P450cam·Pdx complex determined from the isothermal titration and suggested that the recognition mechanism between P450cam and Pdx is similar to that of the antigen·antibody complex formation. A subsequent study performed by Furukawa and Morishima (20Furukawa Y. Morishima I. J. Biol. Chem. 2001; 276: 12983-12990Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) examined the osmotic pressure dependence of the association between P450cam and Pdx and revealed that water molecules participate in the complex formation and cause the negative entropy change upon the complexation of P450cam with Pdx.Other important feature of the P450cam·Pdx complex is that Pdx induces conformational changes of P450cam upon the complex formation (21Unno M. Christian J.F. Benson D.E. Gerber N.C. Sligar S.G. Champion P.M. J. Am. Chem. Soc. 1997; 119: 6614-6620Crossref Scopus (75) Google Scholar, 22Makino R. Iizuka T. Sakaguchi K. Ishimura Y. Nozaki M. Yamamoto S. Ishimura Y. Goon M.J. Ernster L. Estabrook R.W. Oxygenases and Oxygen Metabolism. Academic Press, New York1982: 467-477Google Scholar, 23Unno M. Christian J.F. Sjodin T. Benson D.E. Macdonald I.D. Sligar S.G. Champion P.M. J. Biol. 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Champion P.M. J. Am. Chem. Soc. 1997; 119: 6614-6620Crossref Scopus (75) Google Scholar, 24Lipscomb J.D. Biochemistry. 1980; 19: 3590-3599Crossref PubMed Scopus (159) Google Scholar). Associated with the change of the spin-state, the heme-iron axial ligand (Fe–S) stretching mode is upshifted by 3 cm–1 (21Unno M. Christian J.F. Benson D.E. Gerber N.C. Sligar S.G. Champion P.M. J. Am. Chem. Soc. 1997; 119: 6614-6620Crossref Scopus (75) Google Scholar). In the ferrous-CO form, the Fe–CO and FeC–O stretching modes indicate 2 cm–1 upshift and 8 cm–1 downshift, respectively, by the binding of reduced Pdx, respectively (29Makino R. Iizuka T. Ishimura Y. Uno T. Nishimura Y. Tsuboi M. Proceedings of the Ninth International Conference on Raman Spectroscopy. The Chemical Society of Japan, Tokyo1984: 492-493Google Scholar). Recent multidimensional NMR study on the complex of P450cam with Pdx showed that the binding of Pdx structurally perturbs the several regions involving the substrate access channel in P450cam (28Pochapsky S.S. Pochapsky T.C. Wei J.W. Biochemistry. 2003; 42: 5649-5656Crossref PubMed Scopus (76) Google Scholar).These structural changes of P450cam upon the binding of Pdx are supposed to be essential for the enzymatic activity of P450cam. Shimada et al. (30Shimada H. Nagano S. Ariga Y. Unno M. Egawa T. Hishiki T. Ishimura Y. Masuya F. Obata T. Hori H. J. Biol. Chem. 1999; 274: 9363-9369Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 31Shimada H. Nagano S. Hori H. Ishimura Y. J. Inorg. Biochem. 2001; 83: 255-260Crossref PubMed Scopus (34) Google Scholar) found that the mutations at the putative Pdx binding site, Arg-109 or Arg-112, inhibit the conformational changes in P450cam and suppress the hydroxylation activity to 1–500 μm/min/μm heme corresponding to 1/1000 to 1/3 of that for the wild-type enzyme (12Unno M. Shimada H. Toba Y. Makino R. Ishimura Y. J. Biol. Chem. 1996; 271: 17869-17874Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 31Shimada H. Nagano S. Hori H. Ishimura Y. J. Inorg. Biochem. 2001; 83: 255-260Crossref PubMed Scopus (34) Google Scholar). Although the Pdx-induced structural changes of P450cam have been supposed to be crucial for the P450cam catalysis, it is still unclear how the conformational changes in the active site of P450cam promote the turnover reaction. It is strongly desirable to identify the detailed structural changes of P450cam upon the binding of Pdx.To clarify the conformational changes by the binding of Pdx to P450cam and discuss the functional significance on the catalytic oxygenation reaction, we characterized the Pdx-induced conformational changes of P450cam by using NMR spectroscopy. In the diamagnetic ferrous-CO form of P450cam, the ring-current of the porphyrin separates several resonances of the protons near the heme-iron from the crowded signal region between 0 and 10 ppm (32Dalvit C. Ho C. Biochemistry. 1985; 24: 3398-3407Crossref PubMed Scopus (58) Google Scholar, 33Berzinis A.P. Traylor T.G. Biochem. Biophys. Res. Commun. 1979; 1: 229-235Crossref Scopus (19) Google Scholar, 34Mabbutt B.C. Wright P.E. Biochim. Biophys. Acta. 1985; 832: 175-185Crossref PubMed Scopus (70) Google Scholar). In fact, Mouro et al. (35Mouro C. Bondon A. Simonneaux G. Jung C. FEBS Lett. 1997; 414: 203-208Crossref PubMed Scopus (10) Google Scholar) reported that the β-proton of the axial Cys in carbonmonoxy P450cam indicates the resolved peak at –2.76 ppm. However, most of the ring-current-shifted signals of the ferrous-CO form of P450cam have not yet been assigned. Therefore, we first assigned several ring-current-shifted NMR signals by comparing wild-type and mutants of P450cam in the absence and presence of the substrate and substrate analogues. With the help of the two-dimensional NOESY spectrum, we have successfully assigned the signals arising from d-camphor and Thr-252. Based on these signal assignments, we examined the structural effects of the Pdx binding on the heme environmental structure. As a control experiment for the Pdx-induced structural changes, we also measured the NMR spectrum for carbonmonoxy P450cam in the presence of cyt b 5, whose binding site in P450cam is supposed to overlap with that of Pdx. The detailed information on the structural changes of the heme pocket upon the binding of Pdx allowed us to discuss the functional significance of the Pdx-induced structural changes in P450cam.EXPERIMENTAL PROCEDURESExpression and Purification of Proteins—Wild-type P450cam was expressed in a strain of Escherichia coli, BL21, as an inclusion body. Following the procedures described in our previous reports (36Yoshioka S. Takahashi S. Ishimori K. Morishima I. J. Inorg. Biochem. 2000; 81: 141-151Crossref PubMed Scopus (106) Google Scholar, 37Yoshioka S. Takahashi S. Hori H. Ishimori K. Morishima I. Eur. J. Biochem. 2001; 268: 252-259Crossref PubMed Scopus (47) Google Scholar), we performed the heme reconstitution and the purification of P450cam.P450cam mutants (T252A and T252G) were expressed in an E. coli strain, JM109, and purified as previously reported (38Imai M. Shimada H. Watanabe Y. Matsushima-Hibiya Y. Makino R. Koga H. Horiuchi T. Ishimura Y. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7823-7827Crossref PubMed Scopus (351) Google Scholar). Purified proteins with the R/Z value (A 392/A 280) greater than 1.5 were employed in this study. The purified samples were dissolved in 50 mm potassium phosphate buffer at pH 7.4 containing 50 mm KCl and 1 mm d-camphor and stocked at –70 °C. The substrate-free sample was prepared by passing the substrate bound P450cam through a G-25 column equilibrated with 50 mm Tris-HCl buffer at pH 7.4. The buffer of the eluted sample was exchanged to 50 mm potassium phosphate buffer at pH 7.4 containing 50 mm KCl.Construction of the expression vector for the Asp-38 → Asn mutant of Pdx (D38N) was reported by our group (14Aoki M. Ishimori K. Morishima I. Biochim. Biophys. Acta. 1998; 1386: 157-167Crossref PubMed Scopus (40) Google Scholar). Wild-type Pdx and the D38N mutant were expressed in an E. coli strain, RR1, and were purified by the method of Gunsalus and Wagner (39Gunsalus I.C. Wagner G.C. Methods Enzymol. 1978; 52: 166-188Crossref PubMed Scopus (301) Google Scholar) with a minor modification (40Aoki M. Ishimori K. Morishima I. Wada Y. Inorg. Chim. Acta. 1998; 272: 80-88Crossref Google Scholar). The preparation of Pdx used in this study had a A 325 nm to A 280 nm ratio of at least 0.60. The sample was dissolved in 50 mm potassium phosphate buffer at pH 7.4 containing 50 mm KCl and 80 μl/100 ml β-mercaptoethanol. β-Mercaptoethanol was removed from the solution before the NMR measurements to avoid the interference of the complex formation with P450cam.Rat liver cyt b 5, whose C-terminal membrane binding domain was deleted, was used in this study. The expression and purification of cyt b 5 were carried out by using a method described previously (41Beck von Bodman S. Schuler M.A. Jollie D.R. Sligar S.G. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 9443-9447Crossref PubMed Scopus (197) Google Scholar). Purified proteins with the R/Z value (A 412/A 280) greater than 5.5 were employed. The purified samples were dissolved in 50 mm potassium phosphate buffer at pH 7.4 containing 50 mm KCl and 1-mm d-camphor.NMR Measurements—One-dimensional 1H NMR measurements were performed on a BRUKER Avance DRX500 spectrometer at 40 °C. Chemical shifts were referenced to the residual water signal that was calibrated against tetramethylsilane. The WET and PRESAT pulse sequences were used to minimize the water signal (42Ogg R.J. Kingsley P.B. Taylor J.S. J. Magn. Reson. B. 1994; 104: 1-10Crossref PubMed Scopus (597) Google Scholar). One-dimensional NOE experiments with 30–100 ms of the mixing times were performed using the standard pulse sequence (43Kumar A. Ernst R.R. Wutrich K. Biochem. Biophys. Res. Commun. 1980; 95: 1-6Crossref PubMed Scopus (2014) Google Scholar). Two-dimensional NOESY, COSY and TOCSY spectra were recorded with a BRUKER Avance DRX600 equipped with a cryo-cooled probe at 25 °C by using the standard pulse sequence (43Kumar A. Ernst R.R. Wutrich K. Biochem. Biophys. Res. Commun. 1980; 95: 1-6Crossref PubMed Scopus (2014) Google Scholar, 44Rance M. Sørensen O.W. Bodenhausen G. Wagner G. Ernst R.R. Wuthrich K. Biochem. Biophys. Res. Commun. 1983; 117: 479-485Crossref PubMed Scopus (2594) Google Scholar, 45Bax A. Davis D.G. J. Magn. Reson. 1985; 65: 355-360Google Scholar).NMR samples were prepared by the following procedures. Substrate-bound P450cam was dissolved in 10% D2O or 100% D2O containing 50 mm potassium phosphate, 50 mm KCl, and 1-mm d-camphor at pH (pD) 7.4. Substrate-free sample was dissolved in 10% D2O or 100% D2O containing 50 mm potassium phosphate, 50 mm KCl at pH (pD) 7.4. The d-camphor analogue-bound form of P450cam was prepared by use of the same buffer as the substrate-free sample but saturated with one of the d-camphor analogues: norcamphor, adamantanone, or 3-bromocamphor. Concentrations of P450cam in the NMR samples were 0.2 mm and 0.5–1.0 mm for the one-dimensional NMR and one-dimensional NOE/two-dimensional NOESY measurements, respectively. 500 μl of samples was transferred to the NMR tubes, which were capped with a rubber septum. After flushing with argon gas, samples were reduced by the injection of a small aliquot of degassed sodium dithionite solution. CO gas was anaerobically introduced into the NMR tube to prepare the ferrous-CO form of P450cam.Measurement of the Dissociation Constant (Kd) of Reduced Pdx with the Ferrous-CO Form of P450cam—We measured one-dimensional 1H NMR spectra of the ferrous-CO form of P450cam in the presence of various concentrations of Pdxred from 50 μm to 2 mm. The dissociation constant (K d) of the complex between Pdxred and the ferrous-CO form of P450cam was calculated by the shifts of the NMR signals upon the binding of Pdxred. We confirmed that the rate of equilibrium between Pdx-bound P450cam and Pdx-free P450cam was fast in our experimental condition (see “Results” session). In this fast exchange limit, K d can be determined by Equation 1 (46Lian L.-Y. Roberts G.C.K. Roberts G.C.K. NMR of Macromolecules A Practical Approach. IRL Press, New York1993: 153-182Google Scholar), δobs=δ0+δmax-δ0[P450]tot×Kd+[P450]tot+[Pdx]tot-(Kd+[P450]tot+[Pdx]tot)2-4·[P450]tot·[Pdx]tot2(Eq. 1) where δobs denotes the chemical shift of P450cam in the titration experiment. δmax and δ0 represent the chemical shifts of P450cam in the presence and absence of Pdxred, respectively. [P450]tot and [Pdx]tot correspond to the total concentrations of the ferrous-CO form of P450cam and Pdxred in the sample solution, respectively.RESULTSAssignments of NMR Signals for the Ferrous-CO Form of P450cam— Fig. 1 illustrates the NMR spectra for the ferrous-CO forms of P450cam in the absence and presence of the substrate, d-camphor, and substrate analogues, showing several well resolved and ring-current-shifted resonances in the upfield region (35Mouro C. Bondon A. Simonneaux G. Jung C. FEBS Lett. 1997; 414: 203-208Crossref PubMed Scopus (10) Google Scholar). The intensities of the signals in Fig. 1 were unchanged in the deuterated solution (data not shown). In these ring-current-shifted signals, the signal a observed at –2.76 ppm in the presence of d-camphor has already been assigned to the β-proton of the axial cysteine, Cys-357 (35Mouro C. Bondon A. Simonneaux G. Jung C. FEBS Lett. 1997; 414: 203-208Crossref PubMed Scopus (10) Google Scholar). However, other ring-current-shifted signals b, c, and d have not yet been assigned.The most prominent spectral change by the binding of d-camphor (traces A and E) is the increased intensity of the signal b. Based on the intensity of the signal a, the intensity of the signal b in the absence of the substrate (trace A) corresponds to three protons. The intensity of the signal b was increased to six protons by addition of d-camphor (trace E), indicating that a signal of three protons, which is designated as the signal x, overlapped with the signal b in the presence of d-camphor. It should be noted that the increase of the intensity of the signal b was not observed in the presence of the substrate analogue, norcamphor (trace B) or adamantanone (trace C), whereas the addition of 3-bromocamphor (trace D) increased the intensity of the signal b. These observations suggest that the signal x might be assigned to one of the methyl groups, 8- or 9-methyl groups, attached to the C7 position of d-camphor.To further examine the assignments of the signal x, we measured the two-dimensional NMR spectra, NOESY, COSY, and TOCSY, of the ferrous-CO form of P450cam in the presence of d-camphor. Unfortunately, however, we could not obtain the high quality COSY and TOCSY spectra due to the large molecular weight of P450cam (M r = 46,000) (data not shown) (47Wüthrich K. Nat. Struct. Biol. 1998; 5: 492-495Crossref PubMed Scopus (128) Google Scholar). Thus, the assignment of the signal x was validated by examining the NOESY data in relationship to the crystal structure of carbonmonoxy P450cam. Fig. 2 depicts the upfield region of the NOESY spectra for carbonmonoxy P450cam in the presence and absence of d-camphor at 25 °C. Due to the low temperature to avoid the denaturation of the samples during the NOESY measurements, the chemical shifts for the ring-current-shifted signals in the one-dimensional spectra at the top of Fig. 2 were different from those at 40 °C (Fig. 1). In the absence of d-camphor, the signals b (–1.23 ppm), c (–1.14 ppm), and d (–1.04 ppm) were observed. On the other hand, in the d-camphor-bound enzyme, the signal at –0.90 was observed as a resolved signal, and the signals –1.14 and –1.24 ppm were observed as shoulder signals due to the overlapping with the signal at –1.20 ppm. The signal at –1.23 ppm (signal b) in the substrate-free P450cam gave NOE cross-peaks with the signals at 0.07 (b1), 1.56 (b2), 3.96 (b3), 4.38 (b4), and 4.94 ppm (b5) (red spectrum in Fig. 2), which are the same NOE pattern observed for the shoulder peak at –1.24 ppm in the d-camphor-bound enzyme (black spectrum in Fig. 2). This indicates that the signal at –1.24 ppm in the d-camphor-bound enzyme corresponds to the signal b. The signal at –1.14 ppm (signal c) in the substrate-free enzyme exhibited the NOE cross-peaks (c1, c2, c3, and c4) as found for the signal at –1.14 ppm in the d-camphor-bound enzyme, showing that the signal at –1.14 ppm in the d-camphor-bound enzyme is derived from the protons that give the signal c in the substrate-free form. The signal at –1.04 ppm (signal d in red spectrum of Fig. 2) in substrate-free P450cam displayed strong NOE cross-peaks with the signal from the α-meso proton of the heme (9.74 ppm) (d4) and the signals at 1.33 (d1), 4.01 (d2), and 4.19 ppm (d3), and the same NOE pattern was also observed for the signal at –0.90 ppm in the d-camphor-bound enzyme. These results clearly confirm that the remaining signal at –1.20 ppm (signal x in the black spectrum) is derived from the substrate, d-camphor. It should be noted here that the signal x shows NOEs to the 8-methyl-(3.43 ppm) and δ-mesoprotons (10.17 ppm) of the heme (black spectrum in Fig. 2). Because both of the 8-methyl- and δ-mesoprotons of the heme are located near the 9-methyl group of d-camphor in the crystal structure of the ferrous-CO form of P450cam (48Raag R. Poulos T.L. Biochemistry. 1989; 28: 7586-7592Crossref PubMed Scopus (188) Google Scholar) (Fig. 3), we can unambiguously assign the signal x to the 9-methyl group of d-camphor.Fig. 2Two-dimensional NOESY spectra of the ferrous-CO form of P450cam at 25 °C. The spectra of d-camphor-bound wild-type P450cam, substrate-free wild-type P450cam, and the T252A mutant are shown with black, red, and green, respectively. The top panel represents the one-dimensional NMR spectra of carbonmonoxy P450cam. The signal x gives NOE cross-peaks with the signal d, 8-methyl, and δ-meso protons of the heme. The signal d exhibits NOE cross-peaks with the signal x, α-meso proton of the heme. The sample condition is 0.7–1 mm P450cam in 100% D2O of 50 mm potassium phosphate buffer at pD 7.4 containing 50 mm KCl for the substrate-free enzyme, and 1-mm d-camphor for d-camphor-bound wild-type P450cam and the T252A mutant. Mixing time is 100 ms.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig. 3The crystal structure in the heme vicinity of the ferrous-CO form of P450cam (3CPP) (48Raag R. Poulos T.L. Biochemistry. 1989; 28: 7586-7592Crossref PubMed Scopus (188) Google Scholar). A, side view of the heme plane. B, top view of the heme plane. C, numbering systems for the heme and a camphor molecule. Protons were added to the crystal structure of the ferrous-CO form of P450cam (3CPP) by the Discover program of Insight II (MSI). Atoms are shown by green (carbon), blue (nitrogen), red (oxygen), yellow (sulfur), white (proton), and magenta (iron). The heme and its ligand, CO, are represented by purple. This figure was produced by Insight II (MSI).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The two-dimensional NOESY spectrum of the ferrous-CO form of P450cam

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