FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2090309596> ?p ?o ?g. }

• W2090309596 endingPage "42849" @default.
• W2090309596 startingPage "42844" @default.
• W2090309596 abstract "The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi, A., Hishiki, T., Kimata-Ariga, Y., Egawa, T., Suematsu, M., Park, S.-Y., Adachi, S., Shiro, Y., and Ishimura, Y. (2003) Biochemistry 42, 14507–14514). These effects are unique to Pdx because nonphysiological electron donors are unable to support camphor monooxygenation. The accompanying 1H NMR paper (Tosha, T., Yoshioka, S., Ishimori, K., and Morishima, I. (2004) J. Biol. Chem. 279, 42836–42843) shows that the conformation of active site residues, Thr-252 and Cys-357, and the substrate in the ferrous (Fe(II)) CO complex of the L358P mutant mimics that of the wild-type enzyme complexed to Pdx. To explore how these changes are transmitted from the Pdx-binding site to the active site, we have solved the crystal structures of the ferrous and ferrous-CO complex of wild-type and the L358P mutant. Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdx-binding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with non-physiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor. The cytochrome P450cam active site is known to be perturbed by binding to its redox partner, putidaredoxin (Pdx). Pdx binding also enhances the camphor monooxygenation reaction (Nagano, S., Shimada, H., Tarumi, A., Hishiki, T., Kimata-Ariga, Y., Egawa, T., Suematsu, M., Park, S.-Y., Adachi, S., Shiro, Y., and Ishimura, Y. (2003) Biochemistry 42, 14507–14514). These effects are unique to Pdx because nonphysiological electron donors are unable to support camphor monooxygenation. The accompanying 1H NMR paper (Tosha, T., Yoshioka, S., Ishimori, K., and Morishima, I. (2004) J. Biol. Chem. 279, 42836–42843) shows that the conformation of active site residues, Thr-252 and Cys-357, and the substrate in the ferrous (Fe(II)) CO complex of the L358P mutant mimics that of the wild-type enzyme complexed to Pdx. To explore how these changes are transmitted from the Pdx-binding site to the active site, we have solved the crystal structures of the ferrous and ferrous-CO complex of wild-type and the L358P mutant. Comparison of these structures shows that the L358P mutation results in the movement of Arg-112, a residue known to be important for putidaredoxin binding, toward the heme. This change could optimize the Pdx-binding site leading to a higher affinity for Pdx. The mutation also pushes the heme toward the substrate and ligand binding pocket, which relocates the substrate to a position favorable for regio-selective hydroxylation. The camphor is held more firmly in place as indicated by a lower average temperature factor. Residues involved in the catalytically important proton shuttle system in the I helix are also altered by the mutation. Such conformational alterations and the enhanced reactivity of the mutant oxy complex with non-physiological electron donors suggest that Pdx binding optimizes the distal pocket for monooxygenation of camphor. Cytochrome P450cam (P450cam) 1The abbreviations used are: P450cam, cytochrome P450 (CYP101) originally isolated from Pseudomonas putida; P450, cytochrome P450 (CYP); Pdx, putidaredoxin; WT, wild type; P450cam-O2, d-camphorbound ferrous-O2 P450cam; P450cam-CO, d-camphor-bound ferrous-carbon monoxide P450cam. 1The abbreviations used are: P450cam, cytochrome P450 (CYP101) originally isolated from Pseudomonas putida; P450, cytochrome P450 (CYP); Pdx, putidaredoxin; WT, wild type; P450cam-O2, d-camphorbound ferrous-O2 P450cam; P450cam-CO, d-camphor-bound ferrous-carbon monoxide P450cam. (1Katagiri M. Ganguli B.N. Gunsalus I.C. J. Biol. Chem. 1968; 243: 3543-3546Abstract Full Text PDF PubMed Google Scholar, 2Gunsalus I.C. Meeks J.R. Lipscomb J.D. Debrunner P. Munck E. Hayaishi O. Molecular Mechanism of Oxygen Activation. Academic Press, New York1974: 559-613Google Scholar) catalyzes the regio- and stereo-specific hydroxylation of d-camphor. To activate molecular oxygen, P450cam requires two protons and two electrons. Protons are provided from bulk water through a proton shuttle system that includes Asp-251, Thr-252, and water molecules (3Imai 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, 4Martinis S.A. Atkins W.M. Stayton P.S. Sligar S.G. J. Am. Chem. Soc. 1989; 111: 9252-9253Crossref Scopus (252) Google Scholar, 5Gerber N.C. Sligar S.G. J. Am. Chem. Soc. 1992; 114: 8742-8743Crossref Scopus (153) Google Scholar, 6Gerber N.C. Sligar S.G. J. Biol. Chem. 1994; 269: 4260-4266Abstract Full Text PDF PubMed Google Scholar). Electrons are provided by an iron-sulfur protein, putidaredoxin (Pdx). In the electron transfer steps, P450cam forms a complex with reduced Pdx, and the P450cam active site is perturbed as indicated by various spectroscopic studies including NMR (7Pochapsky S.S. Pochapsky T.C. Wei J.W. Biochemistry. 2003; 42: 5649-5656Crossref PubMed Scopus (76) Google Scholar, 8Tosha T. Yoshioka S. Takahashi S. Ishimori K. Shimada H. Morishima I. J. Biol. Chem. 2003; 278: 39809-39821Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), EPR (9Lipscomb J.D. Biochemistry. 1980; 19: 3590-3599Crossref PubMed Scopus (159) Google Scholar, 10Shimada 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), resonance Raman (11Unno 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, 12Sjodin T. Christian J.F. Macdonald I.D. Davydov R. Unno M. Sligar S.G. Hoffman B.M. Champion P.M. Biochemistry. 2001; 40: 6852-6859Crossref PubMed Scopus (70) Google Scholar, 13Unno M. Christian J.F. Sjodin T. Benson D.E. Macdonald I.D. Sligar S.G. Champion P.M. J. Biol. Chem. 2002; 277: 2547-2553Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), and infrared (14Ishimura Y. Makino R. Iizuka T. Shimada H. Sato R. Omura T. Imai Y. Fujii-Kuriyama Y. Cytochrome P450: New Trends. 17. Yamada Science Foundation, Nara1987: 151Google Scholar, 15Nagano S. Shimada H. Tarumi A. Hishiki T. Kimata-Ariga Y. Egawa T. Suematsu M. Park S.-Y. Adachi S. Shiro Y. Ishimura Y. Biochemistry. 2003; 42: 14507-14514Crossref PubMed Scopus (50) Google Scholar). Because nonphysiological redox proteins such as ferredoxin and adrenodoxin cannot support the monooxygenation reaction or induce the conformational changes (7Pochapsky S.S. Pochapsky T.C. Wei J.W. Biochemistry. 2003; 42: 5649-5656Crossref PubMed Scopus (76) Google Scholar, 16Lipscomb J.D. Sligar S.G. Namtvedt M.J. Gunsalus I.C. J. Biol. Chem. 1976; 251: 1116-1124Abstract Full Text PDF PubMed Google Scholar), the Pdx-induced changes in P450cam are thought to be important for the “effector” function of Pdx in addition to its reductase activity. In support of this view, a recent infrared and mutational study (15Nagano S. Shimada H. Tarumi A. Hishiki T. Kimata-Ariga Y. Egawa T. Suematsu M. Park S.-Y. Adachi S. Shiro Y. Ishimura Y. Biochemistry. 2003; 42: 14507-14514Crossref PubMed Scopus (50) Google Scholar) has shown that Pdx-induced conformational changes promote the camphor monooxygenation reaction.Recent NMR studies found that the CO complex of the L358P mutant has very similar spectroscopic property to the WT P450cam-CO in a complex with Pdx (8Tosha T. Yoshioka S. Takahashi S. Ishimori K. Shimada H. Morishima I. J. Biol. Chem. 2003; 278: 39809-39821Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 17Tosha T. Yoshioka S. Ishimori K. Morishima I. J. Biol. Chem. 2004; 279: 42836-42843Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar). This suggests that the L358P mutant is a good model for Pdx-bound P450cam. However, our knowledge on the conformational change from the NMR studies is limited to only two residues, the axial cysteine ligand (Cys-357) and Thr-252 and the substrate. To overcome this limitation and to complement the NMR work, we have solved the crystal structures of the ferrous and ferrous-CO complex of the L358P mutant.EXPERIMENTAL PROCEDURESProtein Purification and Crystallization—WT P450cam and the L358P 2The “WT” and “L358P” P450cam contain the C334A mutation to prevent intermolecular S–S bond formation that blocks crystal growth (40Nickerson D. Wong L.L. Rao Z. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 470-472Crossref PubMed Google Scholar). Comparison between enzymes with and without the C334A mutation revealed no significant change in catalytic and NMR spectroscopic properties. mutant were overexpressed in Escherichia coli and purified as described elsewhere (18Yoshioka S. Takahashi S. Ishimori K. Morishima I. J. Inorg. Biochem. 2000; 81: 141-151Crossref PubMed Scopus (106) Google Scholar). The CO-bound P450cam structure was first solved by Raag and Poulos (19Raag R. Poulos T.L. Biochemistry. 1989; 28: 7586-7592Crossref PubMed Scopus (188) Google Scholar) by using the initial P212121 crystal form used to solve the P450cam structure. For a more accurate direct comparison between the WT and L358P structures, the ferrous and the ferrous-CO-bound structures for both WT P450cam and the mutant were determined in the P21 crystal form, thus minimizing differences because of crystal lattice and packing effects. Single crystals of WT P450cam and the mutant were obtained by the method described previously (20Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1199) Google Scholar) for WT crystals in the same P21 space group with minor modifications. Crystals of the ferric form were grown at 15 °C. The precipitant solution was 50 mm Tris-HCl buffer, pH 7.4, containing 0.1–0.4 m KCl, 25–32% polyethylene glycol 4,000, and 0.7–1.0 mmd-camphor. The initial droplets contained 1–2 μl of protein solution at the concentration of 20 mg/ml, and 1 μl of the precipitant solution was equilibrated against a reservoir containing 500 μl of the precipitant solution. A single crystal in the ferric form was transferred to a vial containing 15% glycerol as a cryoprotectant. The crystal was anaerobically reduced with 10 mm sodium dithionite for 10 min under anaerobic conditions. Reduction of the ferric enzyme crystals was independently confirmed by measuring the visible absorption spectra of the crystals treated with the procedure described above (spectra not shown). After reduction, CO was passed through the vial for 3 min. The crystal was kept under a CO atmosphere for 1–2 h and then flash-cooled in liquid nitrogen.Data Collection and Processing—All data were collected using an R-Axis IV imaging plate detector (Rigaku) and rotating anode generator equipped with Osmic optics. Crystals were maintained at approximately –160 °C in a cryostream. Data were indexed, integrated, and scaled with HKL2000 (21Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38368) Google Scholar). All crystals belonged to space group P21. Cell dimensions and other data collection statistics are listed in Table I.Table ICrystallographic and refinement statisticsData setFerrous CO-bound L358PFerrous L358PFerrous CO-bound WTFerrous WTUnit cell (Å, °)a = 57.11a = 57.01a = 67.03a = 66.99b = 59.30b = 101.17b = 62.23b = 62.39c = 57.41c = 72.92c = 94.92c = 94.97β = 104.37β = 108.30β = 90.48β = 89.57Space groupP21P21P21P21Resolution range (Å)50-1.8050-1.9050-1.8050-1.90Reflections (observed/unique)171,586/33,098230,635/61,711293,505/72,331212,397/60,650Rmerge (%)aRmerge = Σ|Ii – 〈I〉|/Σ|Ii|, where Ii is the intensity of an observation, and 〈I〉 is the mean value for that reflection, and the summations are overall reflections.,bValues for the highest resolution shell are in parentheses.6.0 (37.6)7.6 (39.1)5.2 (26.1)7.4 (46.0)〈I/σ (I)〉bValues for the highest resolution shell are in parentheses.30.1 (2.6)19.5 (2.4)30.0 (4.4)17.2 (2.2)CompletenessbValues for the highest resolution shell are in parentheses. (%)95.6 (82.3)99.9 (100.0)99.4 (98.1)97.8 (95.8)R/Rfree (%)cR factor = Σh‖Fo(h)| – |Fc(h)‖/Σh| Fo(h)|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. Rfree factor was calculated with 5% of the data.18.9/21.619.9/23.618.5/20.718.7/21.2r.m.s.d. bond length (Å)dr.m.s.d. indicates root mean square difference.0.0060.0060.0050.006r.m.s.d. bond angle (°)1.301.271.321.30No. water molecules296537607510a Rmerge = Σ|Ii – 〈I〉|/Σ|Ii|, where Ii is the intensity of an observation, and 〈I〉 is the mean value for that reflection, and the summations are overall reflections.b Values for the highest resolution shell are in parentheses.c R factor = Σh‖Fo(h)| – |Fc(h)‖/Σh| Fo(h)|, where Fo and Fc are the observed and calculated structure factor amplitudes, respectively. Rfree factor was calculated with 5% of the data.d r.m.s.d. indicates root mean square difference. Open table in a new tab Molecular Replacement, Model Building, and Refinement—Initial models for the ferrous or ferrous-CO-bound forms of the mutant and WT were obtained by molecular replacement with MOLREP (22Vagin A. Teplyakov A. J. Appl. Crystallogr. 1997; 30: 1022-1025Crossref Scopus (4120) Google Scholar) or BEAST (23Read R.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 2001; 57: 1373-1382Crossref PubMed Scopus (777) Google Scholar) by using ferric WT P450cam (Protein Data Bank code 1DZ4 (20Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1199) Google Scholar)) as a search model. The model was built and adjusted by using the graphic program O (24Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (13004) Google Scholar). Refinement was carried out using CNS (25Brunger A.T. Adams P.D. Clore G.M. DeLano W.L. Gros P. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16930) Google Scholar) with maximum likelihood refinement. Five percent of the reflections were set aside for calculation of free R, and the same set of the test reflections was maintained throughout the refinement. Refinement statistics are listed in Table I.After a few cycles of energy minimization and individual temperature factor refinement, the Fo – Fc map clearly showed the heme-bound CO ligand in the L358P mutant that has one molecule in the asymmetric unit (Fig. 1A). However, CO in the WT enzyme was well defined in only one of the two molecules in the asymmetric unit (Fig. 1B). For the second molecule, the Fo – Fcomit map showed a triangularly shaped broad distribution of electron density around the 6th coordination site (Fig. 1C). The electron density distribution was reproducible for crystals from other preparations. The observed heterogeneity in the second molecule in the asymmetric unit is probably because of multiple conformational states and/or a mix of redox states with incomplete CO binding. This appears to be a characteristic of this crystal form because P450cam-O2 crystals also exhibited clearly defined electron density for only one of the molecules in the asymmetric unit (20Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1199) Google Scholar). The same is true for the cyanide complex (26Fedorov R. Ghosh D.K. Schlichting I. Arch. Biochem. Biophys. 2003; 409: 25-31Crossref PubMed Scopus (56) Google Scholar). We used the structure of the first molecule of WT-CO crystal (Fig. 1B) for comparison with that for L358P-CO.RESULTS AND DISCUSSIONL358P Mutation-induced Conformational Changes—The accompanying paper (17Tosha T. Yoshioka S. Ishimori K. Morishima I. J. Biol. Chem. 2004; 279: 42836-42843Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar) shows that the CO complex of the L358P mutant has very similar NMR and vibrational spectral patterns to those of the Pdx-bound WT enzyme. Furthermore, because the vibrational spectroscopic behavior of the CO complex upon Pdx binding parallels that of the oxy complex, structural information gained from the CO complex can serve as a model for changes in structure upon formation of the oxy complex.Leu-358 follows the Cys-357 heme ligand. Although the geometry between the Cys-357 sulfur and Leu-358 peptide NH is not optimal for hydrogen bonding, the 3.6-Å distance is close enough for the partial positive charge on the peptide NH to attenuate the negative charge on the thiolate sulfur. The L358P mutation eliminates such electrostatic stabilization of the Cys-357 thiolate sulfur, which should enhance electron donation from the axial Cys to the heme iron. The enhanced electron donation was confirmed by a slight lowering of 36 mV in the mutant compared with that of WT enzyme (18Yoshioka S. Takahashi S. Ishimori K. Morishima I. J. Inorg. Biochem. 2000; 81: 141-151Crossref PubMed Scopus (106) Google Scholar, 27Yoshioka S. Tosha T. Takahashi S. Ishimori K. Hori H. Morishima I. J. Am. Chem. Soc. 2002; 124: 14571-14579Crossref PubMed Scopus (91) Google Scholar). Vibrational spectroscopic studies on the CO, NO, and oxy complexes have shown that Pdx binding enhances electron donation from the axial Cys, thereby promoting O–O bond cleavage (11Unno 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, 12Sjodin T. Christian J.F. Macdonald I.D. Davydov R. Unno M. Sligar S.G. Hoffman B.M. Champion P.M. Biochemistry. 2001; 40: 6852-6859Crossref PubMed Scopus (70) Google Scholar, 13Unno M. Christian J.F. Sjodin T. Benson D.E. Macdonald I.D. Sligar S.G. Champion P.M. J. Biol. Chem. 2002; 277: 2547-2553Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 15Nagano S. Shimada H. Tarumi A. Hishiki T. Kimata-Ariga Y. Egawa T. Suematsu M. Park S.-Y. Adachi S. Shiro Y. Ishimura Y. Biochemistry. 2003; 42: 14507-14514Crossref PubMed Scopus (50) Google Scholar).Leu-358 makes a number of other nonbonded contacts with the heme pyrrole C-ring and neighboring residues such as Arg-112, Ala-115, and Asn-116 in the C helix. As depicted in Fig. 2, replacement of the Leu with Pro enables the C-terminal side of the C helix to move toward the proximal ligand. Most notably the guanidinium group of Arg-112 relocates about 1 Å closer to Pro-358 and the heme. As discussed below, this is consistent with NMR studies by Pochapsky et al. 3They also reported that the B′, E, and F helix regions were perturbed by the Pdx binding. However, we observed no significant structural changes in this region. (7Pochapsky S.S. Pochapsky T.C. Wei J.W. Biochemistry. 2003; 42: 5649-5656Crossref PubMed Scopus (76) Google Scholar) and may explain the high affinity of the mutant for reduced Pdx.Fig. 2Superimposed structures of L358P and WT P450cam in the ferrous-CO-bound forms. Carbon atoms and helix of L358P and WT are shown in cyan and yellow, respectively. Oxygen, nitrogen, sulfur, and iron atoms are shown in red, blue, orange, and dark pink, respectively. Dotted lines show electrostatic interaction between the thiolate of Cys-357 and the amide N atom of Leu-358. Open arrowheads indicate the position of the heme pyrrole rings A–C. Conformation A (see Fig. 3 and text) for Asp-251 to Thr-252 was omitted from the model for clarity. The two structures were aligned with C-α atoms by using LSQMAN (39Kleywegt G.J. Jones T.A. Structure (Lond.). 1995; 3: 535-540Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar).View Large Image Figure ViewerDownload (PPT)Because of steric crowding, the rigid Pro-358 side chain also forces a 0.4-Å movement of the heme pyrrole ring C away from the Pro-358 side chain as well as movement of the 357–359 backbone away from the heme (Fig. 2). Heme rings A and B move toward the proximal side by 0.3–0.4 Å. These changes slightly increase heme ruffling. Similar ruffling has been observed for the mutant P450cam where Arg-112 was substituted with Lys (15Nagano S. Shimada H. Tarumi A. Hishiki T. Kimata-Ariga Y. Egawa T. Suematsu M. Park S.-Y. Adachi S. Shiro Y. Ishimura Y. Biochemistry. 2003; 42: 14507-14514Crossref PubMed Scopus (50) Google Scholar). Most interestingly, a Pro at the position corresponding to 358 in P450cam has been found in a number of P450 enzymes such as CYP121 (28Leys D. Mowat C.G. McLean K.J. Richmond A. Chapman S.K. Walkinshaw M.D. Munro A.W. J. Biol. Chem. 2003; 278: 5141-5147Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar) and CYP128 (29Cole S.T. Brosch R. Parkhill J. Garnier T. Churcher C. Harris D. Gordon S.V. Eiglmeier K. Gas S. Barry III, C.E. Tekaia F. Badcock K. Basham D. Brown D. Chillingworth T. Connor R. Davies R. Devlin K. Feltwell T. Gentles S. Hamlin N. Holroyd S. Hornsby T. Jagels K. Krogh A. McLean J. Moule S. Murphy L. Oliver K. Osborne J. Quail M.A. Rajandream M.-A. Rogers J. Rutter S. Seeger K. Skelton J. Squares R. Squares S. Sulston J.E. Taylor K. Whitehead S. Barrell B.G. Nature. 1998; 393: 537-544Crossref PubMed Scopus (6450) Google Scholar) from Mycobacterium tuberculosis, CYP7A1 from rabbit (30Twisk J. Hoekman M.F. Mager W.H. Moorman A.F. de Boer P.A. Scheja L. Princen H.M. Gebhardt R. J. Clin. Investig. 1995; 95: 1235-1243Crossref PubMed Scopus (60) Google Scholar), and CYP104 from Agrobacterium tumefaciens (31Kanemoto R.H. Powell A.T. Akiyoshi D.E. Regier D.A. Kerstetter R.A. Nester E.W. Hawes M.C. Gordon M.P. J. Bacteriol. 1989; 171: 2506-2512Crossref PubMed Google Scholar). The crystal structure of CYP121 also shows a ruffled heme conformation because of steric crowding by the Pro ring (28Leys D. Mowat C.G. McLean K.J. Richmond A. Chapman S.K. Walkinshaw M.D. Munro A.W. J. Biol. Chem. 2003; 278: 5141-5147Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar). Because the plausible Pdx-binding site includes Arg-112, a critical residue for the electron transfer reaction and conformational change, it is reasonable to propose that Pdx binding to WT P450cam also pushes the C-ring up as in both the L358P and R112K mutants as well as CYP121. Although the relevance of such ruffling to P450 function remains unclear at present, a ruffled heme conformation could be important in the oxy-P450cam-Pdx complex because spectroscopic studies on metalloporphyrins have shown that ruffling decreases porphyrin π-metal dπ interactions and provides smaller inner sphere reorganization energies and thus enhances electron transfer rates (32Sun H.R. Smirnov V.V. DiMagno S.G. Inorg. Chem. 2003; 42: 6032-6040Crossref PubMed Scopus (36) Google Scholar).To clarify the effect of CO binding on the conformational change, we also determined structures in the ferrous state (structure not shown). The differences between the WT and mutant structures in the proximal pocket of the ferrous proteins are the same as found in the ferrous-CO structure. Therefore, proximal side changes are due to the mutation itself and not a combination of the mutation with CO binding. In sharp contrast, there is no significant difference in the distal pocket between WT and the mutant when the ferrous structures are compared. Therefore, differences in the distal pocket discussed in the next section are due to CO binding in combination with the mutation and not simply the mutation.In combination with the steric effect of CO binding, movement of the heme away from Pro-358 and toward the distal pocket where substrate binds leads to small but significant changes in the distal pocket (Fig. 2). Most notable is a repositioning of the substrate relative to the heme which is best described as a slight rotation about the C-1 atom, which results in a maximum movement of about 0.6 Å in the C-3, C-4, and C-5 atoms. The neighboring groups that contact the substrate like Leu-244 shift away from the distal pocket to allow the reorientation of the substrate.The conformation of the CO ligand in WT P450cam is very similar to that found in the CO-bound form structure reported previously (19Raag R. Poulos T.L. Biochemistry. 1989; 28: 7586-7592Crossref PubMed Scopus (188) Google Scholar). In this earlier work and the current study, the ligand in WT P450cam lies off the heme normal, bent away from the bound substrate, and directed toward Gly-248 in the I helix. The tilt angle between the heme normal and Fe–C bond and bend angle for the Fe–C–O bond are 7 and 151°, respectively, in the WT structure. The CO ligand of L358P also is positioned off of the heme normal and points away from the substrate with an Fe–C tilt of 15° and a Fe–C–O bend of 156°. However, because of the repositioning of the substrate in the L358P mutant compared with WT, the terminal O atom of the ligand moves closer to Thr-252 by 0.7 Å, which is similar to the WT O2 or CN– complexes (33La Mar G.N. Satterlee J.D. De Ropp J.S. Kadish K.M. Smith K.M. Guilard R. The Porphyrin Handbook. 5. Academic Press, San Diego1999: 185-298Google Scholar).In the mutant CO complex, Fo – Fc electron density maps clearly indicate that Asp-251 and Thr-252 have two alternate conformations that have been designated conformers A and B (Fig. 3). In conformer A, the peptide carbonyl of the Asp-251 peptide points toward the substrate, whereas Thr-252 is slightly further away from the substrate. Associated with conformation A are two new water molecules. However, we do not observe multiple conformations nor are the two new water molecules present in the WT-CO complex. This indicates that the two new waters are associated with conformer A of Asp-251 to Thr-252 because of the mutation and not CO binding.Fig. 3The alternate conformations of Asp-251 to Thr-252. The conformation B of Asp-251 and Thr-252, the substrate, and the heme are shown as yellow sticks. The alternate conformation A, which is also found in O- and CN–2 -bound P450cam (20Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1199) Google Scholar, 26Fedorov R. Ghosh D.K. Schlichting I. Arch. Biochem. Biophys. 2003; 409: 25-31Crossref PubMed Scopus (56) Google Scholar), is illustrated as a green stick model. Two water molecules associated with conformation A are shown as green spheres. The simulated annealing Fo – Fc omit maps (conformation A was omitted from the model and 1.0 occupancy was assigned to conformation B) at 2.5- and –2.5-σ contour levels are shown in cyan and red, respectively.View Large Image Figure ViewerDownload (PPT)Conformer A of Asp-251 to Thr-252 was also observed in the O2 or CN–-bound forms, and most interestingly, both ligands have very similar bent conformations even though CN– favors a linear Fe-C-N angle (33La Mar G.N. Satterlee J.D. De Ropp J.S. Kadish K.M. Smith K.M. Guilard R. The Porphyrin Handbook. 5. Academic Press, San Diego1999: 185-298Google Scholar). Electrostatic interactions between the distal ligand and Thr-252 could be a key factor in the observed alternate conformations of Thr-252 and Asp-251. A CN– ligand has a negative charge formally localized at the N atom. Although an O2 molecule is neutral, the oxy complex is usually considered to be in equilibrium between the Fe(II)-O-O and Fe(III)-O-O– resonance forms, or at the very least, the significant negative charge is delocalized on the distal O atom of O2 (34Sharrock M. Debrunner P.G. Schulz C. Lipscomb J.D. Marshall V. Gunsalus I.C. Biochim. Biophys. Acta. 1976; 420: 8-26Crossref PubMed Scopus (122) Google Scholar). Electrostatic interaction between the negatively charged distal ligand and O-γH of Thr-252 could be responsible for the conformation A observed in the O and CN–2 complexes (20Schlichting I. Berendzen J. Chu K. Stock A.M. Maves S.A. Benson D.E. Sweet R.M. Ringe D. Petsko G.A. Sligar S.G. Science. 2000; 287: 1615-1622Crossref PubMed Scopus (1199) Google Scholar, 26Fedorov R. Ghosh D.K. Schlichting I. Arch. Biochem. Biophys. 2003; 409: 25-31Crossref PubMed Scopus (56) Google Scholar). Because a neutral CO ligand does not have such an electrostatic interaction, the conversion from conformer B to A does not occur in the WT-CO complex. However, the A and B conformations are observed in the L358P-CO complex. In this case, the camphor has moved in the mutant closer to the CO compared with WT that forces the oxygen atom in CO to orient toward Thr-252. Therefore, Thr-252 must adopt the A conformation to avoid steric clashes with the ligand and to make electrostatic interaction between the O-γ atom and the ligand.In summary, the mutation causes Arg-112 to move" @default.
• W2090309596 created "2016-06-24" @default.
• W2090309596 creator A5015255703 @default.
• W2090309596 creator A5015794500 @default.
• W2090309596 creator A5023418539 @default.
• W2090309596 creator A5038153411 @default.
• W2090309596 creator A5066417397 @default.
• W2090309596 date "2004-10-01" @default.
• W2090309596 modified "2023-10-18" @default.
• W2090309596 title "Crystal Structure of the Cytochrome P450cam Mutant That Exhibits the Same Spectral Perturbations Induced by Putidaredoxin Binding" @default.
• W2090309596 cites W1529636312 @default.
• W2090309596 cites W1539796472 @default.
• W2090309596 cites W1565067179 @default.
• W2090309596 cites W1603225698 @default.
• W2090309596 cites W1683114625 @default.
• W2090309596 cites W1969513668 @default.
• W2090309596 cites W1972864845 @default.
• W2090309596 cites W1975716269 @default.
• W2090309596 cites W1976008991 @default.
• W2090309596 cites W1979975835 @default.
• W2090309596 cites W1986949928 @default.
• W2090309596 cites W1995017064 @default.
• W2090309596 cites W1997624109 @default.
• W2090309596 cites W2001641653 @default.
• W2090309596 cites W2005083492 @default.
• W2090309596 cites W2011565603 @default.
• W2090309596 cites W2013083986 @default.
• W2090309596 cites W2027003138 @default.
• W2090309596 cites W2032124099 @default.
• W2090309596 cites W2039034683 @default.
• W2090309596 cites W2039105249 @default.
• W2090309596 cites W2050182391 @default.
• W2090309596 cites W2055624255 @default.
• W2090309596 cites W2060107894 @default.
• W2090309596 cites W2069097530 @default.
• W2090309596 cites W2070648332 @default.
• W2090309596 cites W2074621239 @default.
• W2090309596 cites W2083592891 @default.
• W2090309596 cites W2084557052 @default.
• W2090309596 cites W2091733070 @default.
• W2090309596 cites W2097493124 @default.
• W2090309596 cites W2100363956 @default.
• W2090309596 cites W2105216512 @default.
• W2090309596 cites W2132929478 @default.
• W2090309596 cites W2144860443 @default.
• W2090309596 cites W2158023121 @default.
• W2090309596 cites W2160695434 @default.
• W2090309596 cites W2326262377 @default.
• W2090309596 doi "https://doi.org/10.1074/jbc.m404217200" @default.
• W2090309596 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/15269210" @default.
• W2090309596 hasPublicationYear "2004" @default.
• W2090309596 type Work @default.
• W2090309596 sameAs 2090309596 @default.
• W2090309596 citedByCount "54" @default.
• W2090309596 countsByYear W20903095962012 @default.
• W2090309596 countsByYear W20903095962013 @default.
• W2090309596 countsByYear W20903095962014 @default.
• W2090309596 countsByYear W20903095962015 @default.
• W2090309596 countsByYear W20903095962017 @default.
• W2090309596 countsByYear W20903095962018 @default.
• W2090309596 countsByYear W20903095962019 @default.
• W2090309596 countsByYear W20903095962020 @default.
• W2090309596 countsByYear W20903095962021 @default.
• W2090309596 countsByYear W20903095962022 @default.
• W2090309596 crossrefType "journal-article" @default.
• W2090309596 hasAuthorship W2090309596A5015255703 @default.
• W2090309596 hasAuthorship W2090309596A5015794500 @default.
• W2090309596 hasAuthorship W2090309596A5023418539 @default.
• W2090309596 hasAuthorship W2090309596A5038153411 @default.
• W2090309596 hasAuthorship W2090309596A5066417397 @default.
• W2090309596 hasBestOaLocation W20903095961 @default.
• W2090309596 hasConcept C104317684 @default.
• W2090309596 hasConcept C12554922 @default.
• W2090309596 hasConcept C143065580 @default.
• W2090309596 hasConcept C181199279 @default.
• W2090309596 hasConcept C185592680 @default.
• W2090309596 hasConcept C2780768313 @default.
• W2090309596 hasConcept C55493867 @default.
• W2090309596 hasConcept C8010536 @default.
• W2090309596 hasConcept C86803240 @default.
• W2090309596 hasConceptScore W2090309596C104317684 @default.
• W2090309596 hasConceptScore W2090309596C12554922 @default.
• W2090309596 hasConceptScore W2090309596C143065580 @default.
• W2090309596 hasConceptScore W2090309596C181199279 @default.
• W2090309596 hasConceptScore W2090309596C185592680 @default.
• W2090309596 hasConceptScore W2090309596C2780768313 @default.
• W2090309596 hasConceptScore W2090309596C55493867 @default.
• W2090309596 hasConceptScore W2090309596C8010536 @default.
• W2090309596 hasConceptScore W2090309596C86803240 @default.
• W2090309596 hasIssue "41" @default.
• W2090309596 hasLocation W20903095961 @default.
• W2090309596 hasOpenAccess W2090309596 @default.
• W2090309596 hasPrimaryLocation W20903095961 @default.
• W2090309596 hasRelatedWork W1531601525 @default.
• W2090309596 hasRelatedWork W2319480705 @default.
• W2090309596 hasRelatedWork W2384464875 @default.
• W2090309596 hasRelatedWork W2606230654 @default.
• W2090309596 hasRelatedWork W2607424097 @default.

• next