Structure and Function of an NADPH-Cytochrome P450 Oxidoreductase in an Open Conformation Capable of Reducing Cytochrome P450
2009; Elsevier BV; Volume: 284; Issue: 17 Linguagem: Inglês
10.1074/jbc.m807868200
ISSN1083-351X
AutoresDjemel Hamdane, Chuanwu Xia, Sang‐Choul Im, Haoming Zhang, Jung-Ja P. Kim, Lucy Waskell,
Tópico(s)Drug Transport and Resistance Mechanisms
ResumoNADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner. NADPH-cytochrome P450 oxidoreductase (CYPOR) catalyzes the transfer of electrons to all known microsomal cytochromes P450. A CYPOR variant, with a 4-amino acid deletion in the hinge connecting the FMN domain to the rest of the protein, has been crystallized in three remarkably extended conformations. The variant donates an electron to cytochrome P450 at the same rate as the wild-type, when provided with sufficient electrons. Nevertheless, it is defective in its ability to transfer electrons intramolecularly from FAD to FMN. The three extended CYPOR structures demonstrate that, by pivoting on the C terminus of the hinge, the FMN domain of the enzyme undergoes a structural rearrangement that separates it from FAD and exposes the FMN, allowing it to interact with its redox partners. A similar movement most likely occurs in the wild-type enzyme in the course of transferring electrons from FAD to its physiological partner, cytochrome P450. A model of the complex between an open conformation of CYPOR and cytochrome P450 is presented that satisfies mutagenesis constraints. Neither lengthening the linker nor mutating its sequence influenced the activity of CYPOR. It is likely that the analogous linker in other members of the diflavin family functions in a similar manner. NADPH-cytochrome P450 oxidoreductase (CYPOR) 4The abbreviations used are: CYPOR, NADPH-cytochrome P450 oxidoreductase; cyt, cytochrome; FADhq, FAD hydroquinone; FADsq, FAD semiquinone; FADox, oxidized FAD; FMNhq, FMN hydroquinone; FMNsq, FMN semiquinone; FMNox, oxidized FMN; NOS, nitric oxide synthase; r.m.s.d., root mean square deviation; ΔTG, the 2 (ΔThr-236, Gly-237) amino acid deletion mutant; ΔTGEE, the 4 (ΔThr-236, Gly-237, Gly-238, Gly-239) amino acid deletion mutant. is a ∼78-kDa, multidomain, microsomal diflavin protein that shuttles electrons from NADPH → FAD → FMN to members of the ubiquitous cytochrome P450 superfamily (1.Masters B.S. Okita R.T. Pharmacol. Ther. 1980; 9: 227-244Crossref PubMed Scopus (43) Google Scholar, 2.Paine M.J. Scrutton N.S. Munro A.W. Gutierrez A. Robert G.C.K. Wolf C.R. Ortiz de Montellano P.R. Cytochrome P450. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 115-148Crossref Scopus (91) Google Scholar). In humans, the cytochromes P450 (cyt P450) are one of the most important families of proteins involved in the biosynthesis and degradation of a vast number of endogenous compounds and the detoxification and biodegradation of most foreign compounds. CYPOR also donates electrons to heme oxygenase (3.Schacter B.A. Nelson E.B. Marver H.S. Masters B.S. J. Biol. Chem. 1972; 247: 3601-3607Abstract Full Text PDF PubMed Google Scholar), cytochrome b5 (4.Enoch H.G. Strittmatter P. J. Biol. Chem. 1979; 254: 8976-8981Abstract Full Text PDF PubMed Google Scholar), and cytochrome c (5.Horecker B.L. J. Biol. Chem. 1950; 183: 593-605Abstract Full Text PDF Google Scholar). The FAD receives a hydride anion from the obligate two electron donor NADPH and passes the electrons one at a time to FMN. The FMN then donates electrons to the redox partners of CYPOR, again one electron at a time. Cyt P450 accepts electrons at two different steps in its complex reaction cycle. Ferric cyt P450 is reduced to the ferrous protein, and oxyferrous cyt P450 receives the second of the two electrons to form the peroxo (Fe+3OO)2- cyt P450 intermediate (6.Ortiz de Montellano P.R. De Voss J.J. Ortiz de Montellano P.R. Cytochrome P450. 3rd Ed. Kluwer Academic/Plenum Publishers, New York2005: 183-245Crossref Scopus (207) Google Scholar). In vivo, CYPOR cycles between the one- and three-electron reduced forms (7.Iyanagi T. Makino N. Mason H.S. Biochemistry. 1974; 13: 1701-1710Crossref PubMed Scopus (158) Google Scholar, 8.Oprian D.D. Coon M.J. J. Biol. Chem. 1982; 257: 8935-8944Abstract Full Text PDF PubMed Google Scholar). Although the one-electron reduced form is an air-stable, neutral blue semiquinone (FMNox/sq, -110 mV), it is the FMN hydroquinone (FMNsq/hq, -270 mV), not the semiquinone, that donates an electron to its redox partners (8.Oprian D.D. Coon M.J. J. Biol. Chem. 1982; 257: 8935-8944Abstract Full Text PDF PubMed Google Scholar, 9.Vermilion J.L. Coon M.J. J. Biol. Chem. 1978; 253: 8812-8819Abstract Full Text PDF PubMed Google Scholar, 10.Vermilion J.L. Ballou D.P. Massey V. Coon M.J. J. Biol. Chem. 1981; 256: 266-277Abstract Full Text PDF PubMed Google Scholar, 11.Kurzban G.P. Strobel H.W. J. Biol. Chem. 1986; 261: 7824-7830Abstract Full Text PDF PubMed Google Scholar). CYPOR is the prototype of the mammalian diflavin-containing enzyme family, which includes nitric-oxide synthase (12.Bredt D.S. Hwang P.M. Glatt C.E. Lowenstein C. Reed R.R. Snyder S.H. Nature. 1991; 351: 714-718Crossref PubMed Scopus (2171) Google Scholar), methionine synthase reductase (13.Leclerc D. Wilson A. Dumas R. Gafuik C. Song D. Watkins D. Heng H.H. Rommens J.M. Scherer S.W. Rosenblatt D.S. Gravel R.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 3059-3064Crossref PubMed Scopus (352) Google Scholar, 14.Olteanu H. Benerjee R. J. Biol. Chem. 2001; 276: 35558-35563Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar), and a novel reductase expressed in the cytoplasm of certain cancer cells (15.Paine M.J. Garner A.P. Powell D. Sibbald J. Sales M. Pratt N. Smith T. Tew D.G. Wolf C.R. J. Biol. Chem. 2000; 275: 1471-1478Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). CYPOR is also a target for anticancer therapy, because it reductively activates anticancer prodrugs (16.Rooseboom M. Commandeur J.N. Vermeulen N.P. Pharmacol. Rev. 2004; 56: 53-102Crossref PubMed Scopus (436) Google Scholar). CYPOR consists of an N-terminal single α-helical transmembrane anchor (∼6 kDa) responsible for its localization to the endoplasmic reticulum and the soluble cytosolic portion (∼66 kDa) capable of reducing cytochrome c. Crystal structures of the soluble form of the wild-type and several mutant CYPORs are available (17.Wang M. Roberts D.L. Paschke R. Shea T.M. Siler-Masters B.S. Kim J.J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 944: 8411-8416Crossref Scopus (668) Google Scholar, 18.Hubbard P.A. Shen A.L. Paschke R. Kasper C.B. Kim J.J.P. J. Biol. Chem. 2001; 276: 29163-29170Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The first ∼170 amino acids of the soluble domain are highly homologous to flavodoxin and bind FMN (FMN domain), whereas the C-terminal portion of the soluble protein consists of a FAD- and NADPH-binding domain with sequence and structural similarity to ferredoxin-NADP+ oxidoreductase (FAD domain). A connecting domain, possessing a unique sequence and structure, joins the FMN and FAD domains and is partly responsible for the relative orientation of the FMN and FAD domains. In the crystal structure, a convex anionic surface surrounds FMN. In the wild-type crystal structure, the two flavin isoalloxazine rings are in van der Waals contact, poised for efficient interflavin electron transfer (17.Wang M. Roberts D.L. Paschke R. Shea T.M. Siler-Masters B.S. Kim J.J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 944: 8411-8416Crossref Scopus (668) Google Scholar). Based on the juxtaposition of the two flavins, an extrinsic electron transfer rate of ∼1010 s-1 is predicted (19.Page C.C. Moser C.C. Chen X.X. Dutton P.L. Nature. 1999; 402: 47-52Crossref PubMed Scopus (1516) Google Scholar). However, the experimentally observed electron transfer rate between the two flavins is 30–55 s-1 (20.Bhattacharyya A.K. Lipka J.J. Waskell L. Tollin G. Biochemistry. 1991; 30: 759-765Crossref PubMed Scopus (42) Google Scholar, 21.Gutierrez A. Paine M. Wolf C.R. Scrutton N.S. Roberts G.C. Biochemistry. 2002; 41: 4626-4637Crossref PubMed Scopus (80) Google Scholar). This modest rate and slowing of electron transfer in a viscous solvent (75% glycerol) suggest that interflavin electron transfer is likely conformationally gated. Moreover, the “closed” crystal structure, in which the flavins are in contact, is difficult to reconcile with mutagenesis studies that indicate the acidic amino acid residues on the surface near FMN are involved in interacting with cyt P450 (22.Shen A.L. Kasper C.B. J. Biol. Chem. 1995; 270: 27475-27480Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The first structural insight into how cyt P450 might interact with the FMN domain of CYPOR was provided by the crystal structure of a complex between the heme and FMN-containing domains of cyt P450 BM3 (23.Sevrioukova I.F. Li H. Zhang H. Peterson J.A. Poulos T.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1863-1868Crossref PubMed Scopus (461) Google Scholar). In this complex, the methyl groups of FMN are oriented toward the heme on the proximal surface of cyt P450 BM3. Considered together, these three observations, the slow interflavin electron transfer, the mutagenesis data, and the structure of the complex between the heme and FMN domains of cyt P450 BM3, suggest that CYPOR will undergo a large conformational rearrangement in the course of shuttling electrons from NADPH to cyt P450. In addition, crystal structures of various CYPOR variants indicate that the FMN domain is highly mobile with respect to the rest of the molecule (18.Hubbard P.A. Shen A.L. Paschke R. Kasper C.B. Kim J.J.P. J. Biol. Chem. 2001; 276: 29163-29170Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). Consideration of how the reductase would undergo a reorientation to interact with its redox partners led us to hypothesize the existence of a structural element in the reductase that would regulate the conformational changes and the relative dynamic motion of the domains. Our attention focused on the hinge region between the FMN and the connecting domain, because it is often disordered and highly flexible in the crystal structure (supplemental Fig. S1). The length and sequence of the hinge have been altered by site-directed mutagenesis, and the effects of the mutations on the catalytic properties of each mutant have been determined. The results demonstrate that lengthening the linker or altering its sequence do not modify the properties of CYPOR. In contrast, deletion of four amino acids markedly disrupts electron transfer from FAD to FMN, whereas the ability of the FMN domain to donate electrons to cyt P450 remains intact. The hinge deletion variant has been crystallized in three “open” conformations capable of interacting with cyt P450. Determination of CYPOR Activity Using Cytochrome c and Ferricyanide—The ability of CYPOR hinge mutants to reduce cytochrome c and ferricyanide was measured in 270 mm potassium phosphate buffer, pH 7.7, at 30 °C as described previously (24.Shen A.L. Porter T.D. Wilson T.E. Kasper C.B. J. Biol. Chem. 1989; 264: 7584-7589Abstract Full Text PDF PubMed Google Scholar). Details of the assay are provided in the supplemental materials. To ensure that the loss of cytochrome c activity observed with the two (ΔT236, G237) (ΔTG) and four (ΔT236, G237, E238, E239) (ΔTGEE) amino acid deletion mutants was not due to a dissociation of flavins from the CYPOR, the reduction of cytochrome c assays was also measured in the presence of 240 nm free FMN and FAD. The added flavins did not significantly increase the activity of the CYPOR. The concentration of free FMN and FAD was determined using their extinction coefficients (ϵFMN445 nm = 12.5 mm-1cm-1; ϵFAD450 nm = 11.3 mm-1 cm-1) (11.Kurzban G.P. Strobel H.W. J. Biol. Chem. 1986; 261: 7824-7830Abstract Full Text PDF PubMed Google Scholar). The kcat and Km for cytochrome c and NADPH were determined by following the kinetics of cytochrome c reduction at varying concentrations of cytochrome c (65, 28, 18.5, 9.2, and 1.9 μm) and NADPH (60, 30, 15, 9, 3, and 1.2 μm). The kinetic parameters were obtained by fitting the initial rate versus concentration of the substrate using the Michaelis-Menten equation. Reduction of ferricyanide by CYPOR was measured by following the bleaching of the absorbance at 420 nm. Typically, 10 nm CYPOR was preincubated with 500 μm oxidized ferricyanide for 5 min at 30 °C. The reaction was started by the addition of NADPH to a final concentration of 100 μm. The concentration of the reduced ferricyanide was calculated using an extinction coefficient at 420 nm of 1.02 mm-1 cm-1 (11.Kurzban G.P. Strobel H.W. J. Biol. Chem. 1986; 261: 7824-7830Abstract Full Text PDF PubMed Google Scholar). Measurement of Benzphetamine Metabolism by Cyt P450 2B4—Cyt P450 2B4 was expressed and purified as described previously (25.Saribas A.S. Gruenke L. Waskell L. Protein Expr. Purif. 2001; 21: 303-309Crossref PubMed Scopus (44) Google Scholar) (details in supplemental materials). The metabolism of benzphetamine at 30 °C under steady-state conditions was determined by measuring formaldehyde formation using Nash's reagent as previously described (26.Bridges A.G.L. Chang Y.T. Vaksar I.A. Loew G. Waskell L. J. Biol. Chem. 1998; 273: 17036-17049Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). Kinetics of the Reduction of CYPOR by NADPH—The experiments were performed at 25 °C under anaerobic conditions using a High-Tech SF61DX2 stopped-flow spectrophotometer equipped with a temperature-controlled circulating water bath, housed in an anaerobic Belle Technology glove box (Hi-Tech, Salisbury, England) as previously described (8.Oprian D.D. Coon M.J. J. Biol. Chem. 1982; 257: 8935-8944Abstract Full Text PDF PubMed Google Scholar). Additional experimental details are included in the supplemental materials. Determination of the Rate of Reduction of Ferric Cyt P450 2B4 by CYPOR in the Presence and Absence of Benzphetamine—The rate of reduction of ferric cyt P450 2B4 in the presence of CO by wild-type, ΔTG, and ΔTGEE CYPOR was determined as previously described (27.Oprian D.D. Vatsis K.P. Coon M.J. J. Biol. Chem. 1979; 254: 8895-8902Abstract Full Text PDF PubMed Google Scholar). Details are in the supplemental materials. Determination of the Kinetics of Product Formation by Oxyferrous Cyt P450 2B4 in the Presence of Two-electron Reduced Wild-type and Variant CYPOR—The rate of oxidation of cyclohexane and benzphetamine by cyt P450 2B4 in the presence of the two-electron reduced CYPOR was performed at 30 and 15 °C, respectively, using a QFM-400 chemical quench flow apparatus (BioLogic, France) as described (28.Zhang H. Im S.-C. Waskell L. J. Biol. Chem. 2007; 282: 29766-29776Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Briefly, a protein complex between oxidized reductase and ferric cyt P450 was preformed in the presence of dilauroyl l-3-phosphatidyl choline at a molar ratio of 1:1:60 (CYPOR:cyt P450:dilauroyl l-3-phosphatidyl choline). The substrate (cyclohexane or benzphetamine) was added to the protein mixture at a final concentration of 1 mm. Then the preformed cyt P450·CYPOR complex was stoichiometrically reduced with dithionite under anaerobic conditions to the ferrous state for cyt P450 and to the two-electron reduced state for CYPOR. The solution containing the reduced, preformed cyt P450·CYPOR complex was loaded into syringe 1 of the chemical quenched-flow apparatus and rapidly mixed with aerobic buffer ([O2] ≈ 0.32 mm) containing 1 mm of substrate from syringe 2. The reaction was quenched with 1 m NaOH at different times. The samples were collected, and the product in each sample was quantified as described (28.Zhang H. Im S.-C. Waskell L. J. Biol. Chem. 2007; 282: 29766-29776Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Cyclohexanol was measured by a gas-chromatography mass spectrometry assay (Agilent Technologies HP6890/MSO5893). Norbenzphetamine was quantified using a high-pressure liquid chromatography-mass spectrometer assay (ThermoFinnigan TSQ). Crystallization and Data Collection—The Δ56 form of the ΔTGEE mutant (hereafter referred to as the soluble form), in which the first 56 residues of the N terminus, including the membrane anchor of ΔTGEE, were deleted, was constructed and expressed in a manner similar to that of the full-length ΔTGEE protein (details in supplemental materials). The purification procedure (details in supplemental materials) was also similar except that 0.1% Triton X-100 rather than 0.3% Triton X-100 was used, and an affinity column (2′,5′-ADP-Sepharose) was included at the final step. Prior to crystallization setup, 2× and 20× molar excesses of FMN and NADP+, respectively, were added to the protein sample followed by two cycles of concentration/dilution to remove excess cofactors. The protein solution was concentrated to 15 mg/ml in 50 mm HEPES, pH 7.5. Crystals were grown using the hanging drop method by mixing 1.8 μl of the protein solution and 2 μl of reservoir solution (100 mm HEPES, pH 7.5, 150 mm NH4SCN, and 15% polyethylene glycol 5000 monomethylester). Small thin plate-shaped crystals appeared within a week. Crystals were soaked in a cryoprotectant solution (reservoir buffer plus 10% glycerol) for ∼5 s and then flash frozen in liquid nitrogen prior to data collection. A 3.4-Å data set was collected at the SBC 19ID beamline, Advanced Photon Source. Data processing was done with the program HKL2000 (29.Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref PubMed Scopus (38573) Google Scholar). The crystals belong to the space group P21 with unit cell dimensions of a = 109.1Å, b = 93.1Å, and c = 125.7 Å and β = 100.0° and contained three molecules of the ΔTGEE mutant per asymmetric unit. Structure Determination—Molecular replacement methods were employed for phase determination. Not surprisingly, no reasonable solutions were obtained using either the program, MOLREP in CCP4 (CCP4, Programs for protein crystallography (30.Collaborative Computational Project Number 4Acta Crystallogr. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19770) Google Scholar)) or Phaser (31.McCoy A.J. Grosse-Kuntstleve R.W. Adams P.D. Winn M.D. Storoni L.C. Read R.J. J. Appl. Crystallogr. 2007; 40: 658-674Crossref PubMed Scopus (14567) Google Scholar), when the wild-type rat CYPOR structure (PDB ID, 1AMO) was used as the search model. However, when separate domains of CYPOR, i.e. the FAD domain and the FMN domain, were used as search models, Phaser successfully located three FAD domains and two FMN domains sequentially with the rotation function Z score/translation function Z scores/log likelihood gain of 18.5/15.4/367, 15.7/37.6/1294, 12.7/42.9/2392, 7.8/40.4/3211, and 2.6/15.1/4688, respectively, with an R value of 40.9%. Because all the cofactors were excluded from the search models, the strong electron densities for FAD and FMN at expected positions confirmed the correctness of the solution. The third FMN domain could not be located at this stage, although there was a large enough empty space for an FMN domain in the crystal packing. When four cofactors (three FAD and one FMN) were added to the model, then the linker region between the two flavin domains of the second molecule was clearly visible in the |Fo| - |Fc| difference Fourier map (Fig. 2). In addition, the adenosine pyrophosphate portion of NADP+ in all three FAD domains could be identified. Further refinements were carried out using the program CNS (32.Brunger A.T. Nat. Protocols. 2007; 2: 2728-2733Crossref PubMed Scopus (1131) Google Scholar) and manual model adjustments with COOT (33.Emsley P. Cowtan K. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23389) Google Scholar), yielding R-factors of 33.3% and 25.1% for R-free and R-crystal, respectively. During the refinement, tight non-crystallographic symmetry restraints for the three FAD domains and two FMN domains were used until the last cycle of refinement, which gave final R-factors of 27.3% and 21.4% for R-free and R-crystal, respectively. At this stage, all three FAD domains are well defined. However, only one FMN domain is clearly defined and the B-factors of the β-sheets and loop areas of the second FMN domain were extremely high (∼200 Å2). Therefore, all but 78 residues, mostly corresponding to α helices, were excluded from the subsequent refinement. At this stage, some patches of densities in the |Fo| - |Fc| map were visible extending from the third hinge that could be assigned to the α helices F and B of the third FMN domain, and thus the third FMN domain could be modeled. However, this FMN domain was not included in the refinement. The final R-factors were 27.9% for R-free and 21.9% for R-crystal. Data collection and the refinement statistics are given in Table 1.TABLE 1Data collection and refinement statisticsSampleΔTGEEData collectionResolution (Å)50-3.4 (3.52-3.40)aNumbers in parentheses are for the highest resolution shellTotal measured reflections142,069Unique reflections33,940Completeness (%)97.4 (83.3)Redundancy4.2 (2.4)I/σ(I)11.0 (1.1)Unit cell dimensions a, b, c (Å); β (°)109.1, 93.0, 125.7; 100.0Space groupP 21Rsym0.134 (0.652)No. of monomers in an asymmetric unit3Vm (Da/Å3)3.0RefinementResolution (Å)30-3.4Rcrystal/Rfree0.219/0.279r.m.s.d. bond (Å)/angle (°)0.009/1.5B-factor analysis (Å2) Wilson B86.0 Mol A: FAD/FMN domain98.6/95.4 Mol B: FAD/FMN domain93.8/160.0bOnly 78 of a total of 172 residues were included in the refinement Mol C: FAD domain117.9Ramachandran analysis Most favored (%)71.6 Allowed (%)26.0 Generously allowed (%)1.8 Disallowed (%)0.6a Numbers in parentheses are for the highest resolution shellb Only 78 of a total of 172 residues were included in the refinement Open table in a new tab Docking of CYPOR and Cyt P450 2B4—Two docking methods were employed: GRAMM-X (34.Tovchigrenchko A. Vakser I.A. Nucleic Acids Res. 2006; 22: W310-W314Crossref Scopus (585) Google Scholar) and Z-DOCK (35.Wiehe K. Peterson M.W. Pierce B. Mintseris J. Weng Z. Methods Mol. Biol. 2007; 413: 283-314Google Scholar). In the GRAMM-X docking, Mol A was assigned as the receptor molecule and cyt P450 2B4 (PDB code, 1SUO) as the ligand. Glu-208 of Mol A and any four of the nine residues (Arg-122, Lys-126, Arg-133, Phe-135, Met-137, Lys-139, Arg-422, Lys-433, and Arg-443) of cyt P450 were required to be in the interface area. Mutagenesis studies have identified Glu-208 of CYPOR as being involved in the interaction with cyt P450 (22.Shen A.L. Kasper C.B. J. Biol. Chem. 1995; 270: 27475-27480Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). The nine residues of cyt P450 2B4 have been shown by site-directed mutagenesis to be involved in the interaction with CYPOR (26.Bridges A.G.L. Chang Y.T. Vaksar I.A. Loew G. Waskell L. J. Biol. Chem. 1998; 273: 17036-17049Abstract Full Text Full Text PDF PubMed Scopus (259) Google Scholar). In using ZDOCK, again, Glu-208 of Mol A and Arg-133 and Lys-433 of cyt P450 were included as required residues in the contact area. Among the top 10 solutions obtained from each method, the only common solution (fifth in both methods) was selected. No further refinement of the docked structure was attempted. Rationale for Mutations in the 12-Amino Acid Hinge Joining the FMN Domain to the Remainder of the Protein—To determine whether the flexible random-coil hinge joining the FMN to the connecting domain of CYPOR functioned in orienting the FMN and FAD domains of the protein for electron transfer from FAD via FMN to the acceptor protein cyt P450, the hinge was mutated. To identify the boundaries of the hinge a multiple sequence alignment of the 45 known CYPOR sequences and a structural alignment of two CYPOR structures (rat and yeast) were performed (17.Wang M. Roberts D.L. Paschke R. Shea T.M. Siler-Masters B.S. Kim J.J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 944: 8411-8416Crossref Scopus (668) Google Scholar, 18.Hubbard P.A. Shen A.L. Paschke R. Kasper C.B. Kim J.J.P. J. Biol. Chem. 2001; 276: 29163-29170Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 36.Lamb D.C. Kim Y. Yermalitskaya L.V. Yermalitsky V.N. Lepesheva G.I. Kelly S.L. Waterman M.R. Podust L.M. Structure. 2006; 14: 51-56Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar) (supplemental Fig. S1). The alignments indicated the length of the linker was conserved and that it spanned 12 residues from Gly-232 to Arg-243 in the rat protein. Although the sequence identity between rat and yeast reductase is only 25%, the structures were highly conserved (r.m.s.d. for backbone atoms, 1.4 Å) (36.Lamb D.C. Kim Y. Yermalitskaya L.V. Yermalitsky V.N. Lepesheva G.I. Kelly S.L. Waterman M.R. Podust L.M. Structure. 2006; 14: 51-56Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Rat neuronal nitric-oxide synthase (NOS) reductase has an analogous, 24-amino acid flexible hinge, which influences the electron flow through the NOS reductase to an acceptor molecule (37.Haque M.M. Panda K. Tejero J. Aulak S. Fadalla M.A. Mustovich A.T. Stuehr D.J. Proc. Natl. Acad. Sci. U. S. A. 2007; 104: 9254-9259Crossref PubMed Scopus (57) Google Scholar, 38.Garcin E.D. Bruns C.M. Lloyd S.J. Hosfield D.J. Tiso M. Gachhui R. Stuehr D.J. Tainer J.A. Getzoff E.D. J. Biol. Chem. 2004; 279: 37918-37927Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar, 39.Ilagan R.P. Tiso M. Konas D.W. Hermann C. Durra D. Hille R. Stuehr D.J. J. Biol. Chem. 2008; 283: 19603-19615Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). The NOS hinge sequence is not conserved with respect to the hinge of CYPOR. To investigate whether the length of the CYPOR hinge altered electron flow through the reductase and/or modulated the ability to reduce its electron transfer partner, cyt P450, the length of the hinge was increased by two and four alanine residues and shortened by two and four amino acids, using protein engineering techniques. The hinge also contains two charged residues (Glu-238 and Glu-239), which form salt bridges with the guanidinium side chain of Arg-104 in the FMN domain. To test the influence of these salt bridges on the stability of the closed conformation, Glu-238 and Glu-239 were mutated to alanine. In addition, a mutant with four amino acid changes in the middle of the hinge (Thr-236 → Ala, Gly-237 → Ala, Glu-238 → Ala, and Glu-239 → Ala) was constructed to determine if these particular amino acids and the sequence were required for normal function. Activity of the Mutant Reductases with Cyt P450 2B4, Cytochrome c, and Ferricyanide—The ability of the CYPOR variants to support catalysis by the physiological redox partner, full-length cyt P450 2B4, was measured by quantitating the N-demethylation of benzphetamine to formaldehyde (Table 2). The 2- and 4-residue substitution mutants (E238A/E239A and T236A/G237A/E238A/E239A) and the 2- and 4-alanine addition mutants exhibited normal or slightly increased activity. The 2-amino acid deletion mutant, ΔTG 236–237 (hereafter, simply referred to as ΔTG), supported a wild-type rate of catalysis, whereas the activity of the 4-amino acid deletion ΔTGEE 236–239 (hereafter referred to as ΔTGEE) was undetectable.TABLE 2Activity of the wild-type and CYPOR mutants with redox partners The activity of reductases with their redox partners cytochrome c, ferricyanide, and cyt P450 2B4 was determined at 30 °C as described under “Experimental Procedures.” The activity of CYPOR with cyt P450 2B4 is reported in units of nmol of formaldehyde produced/s/nmol of CYPOR.Reductase mutantscyt P450Fe3+ cyt cFe(CN)63-nmol/s/nmol CYPORnmol reduced/s/nmol CYPORWild type0.67 ± 0.0770.6 ± 5.1135 ± 6.4ΔTG-(236–237)0.77 ± 0.08311 ± 0.64170 ± 9ΔTGEE-(236–239)NDaND, not detected0.33 ± 0.026136 ± 3.9E238A/E239A0.95 ± 0.178 ± 6.4141 ± 6.4T236A/G237A/E238A/E239A0.83 ± 0.0877 ± 6.4167 ± 10G237/+AA/E2380.83 ± 0.1109 ± 4137 ± 5G237/+AAAA/E2380.82 ± 0.07106 ± 2.6160 ± 6.5a ND, not detected Open table in a new tab Cytochrome c is not a physiological redox partner for CYPOR. Nevertheless, it is often used to assess the reductase activity because of the simplicity and rapidity of the assay (24.Shen A.L. Porter T.D. Wilson T.E. Kasper C.B. J. Biol. Chem. 1989; 264: 7584-7589Abstract Full Text PDF PubMed Google Scholar). Moreover, there is evidence that the binding site for cyt P450 and cytochrome c, both basic proteins, on the acidic FMN domain of the reductase is either identical or overlapping (40.Tamburini P.P. Schenkman J.B. Mol. Pharmacol. 1986; 30: 178-185PubMed Google Scholar, 41.Davydov D.R. Darovsky B.V. Dedinsky I.R. Kanaeva I.P. Bachmanova G.I. Blinov V.M. Archakov A.L. Arch. Biochem. Biophys. 1992; 297: 304-313Crossr
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