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Probing the Interactions of Putidaredoxin with Redox Partners in Camphor P450 5-Monooxygenase by Mutagenesis of Surface Residues

1997; Elsevier BV; Volume: 272; Issue: 35 Linguagem: Inglês

10.1074/jbc.272.35.21720

1083-351X

Marcia J. Holden, Martin P. Mayhew, David M. Bunk, Adrián E. Roitberg, Vincent L. Vilker,

Analytical Chemistry and Chromatography

The role of surface amino acid residues in the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450cam (CYP101) system was investigated by site-directed mutagenesis. The mutated Pdx genes were expressed inEscherichia coli, and the proteins were purified and studied in vitro. Activity of the complete reconstituted P450cam system was measured, and kinetic parameters were determined. Partial assays were also conducted to determine the effect of the mutations on interactions with each redox partner. Some mutations altered interactions of Pdx with one redox partner but not the other. Other mutations affected interactions with both redox partners, suggesting some overlap in the binding sites on Pdx for putidaredoxin reductase and CYP101. Cysteine 73 of Pdx was identified as important in the interaction of Pdx with putidaredoxin reductase, whereas aspartate 38 serves a critical role in the subunit binding and electron transfer to CYP101. The role of surface amino acid residues in the interaction of putidaredoxin (Pdx) with its redox partners in the cytochrome P450cam (CYP101) system was investigated by site-directed mutagenesis. The mutated Pdx genes were expressed inEscherichia coli, and the proteins were purified and studied in vitro. Activity of the complete reconstituted P450cam system was measured, and kinetic parameters were determined. Partial assays were also conducted to determine the effect of the mutations on interactions with each redox partner. Some mutations altered interactions of Pdx with one redox partner but not the other. Other mutations affected interactions with both redox partners, suggesting some overlap in the binding sites on Pdx for putidaredoxin reductase and CYP101. Cysteine 73 of Pdx was identified as important in the interaction of Pdx with putidaredoxin reductase, whereas aspartate 38 serves a critical role in the subunit binding and electron transfer to CYP101. Multiprotein redox enzyme systems such as methane monooxygenase, cytochrome P450s, and diooxygenases of similar molecular architecture are being investigated as biocatalysts for conversion of organic substrates with no functional groups into oxygen-bearing compounds with high regio- or stereo-selectivity. Maximizing the catalytic efficiency of such systems requires knowledge of the pathways of electron transfer and of the surface regions and amino acid residues involved in the interaction of the redox partner subunits. Cytochrome P450cam (CYP101) has been intensively investigated for over 20 years as a model P450 system (1Mueller E.J. Loida P.J. Sligar S.G. Ortiz de Montellano P.R. Cytochrome P450 Structure, Mechanism, and Biochemistry. Plenum Press, New York1995: 83-124Crossref Google Scholar). This soluble P450 (from Pseudomonas putida) consists of three subunits: putidaredoxin reductase (PdR, 1The abbreviations used are: PdR, putidaredoxin reductase; camphor, (1R)-camphor; 5-exo-hydroxycamphor, (1R)-5-exo-hydroxycamphor; Pdx, putidaredoxin; WT, wild type. Mr ≈ 43,500), putidaredoxin (Pdx,Mr ≈ 11,600), and cytochrome P450cam hydroxylase (CYP101, Mr ≈ 45,000). The genes, camA (PdR), camB (Pdx), andcamC (CYP101) from the cam operon have been cloned and sequenced, and the protein subunits were expressed in individual clones (2Koga H. Rauchfuss B. Gunsalus I.C. Biochem. Biophys. Res. Commun. 1985; 130: 412-417Crossref PubMed Scopus (37) Google Scholar, 3Unger B.P. Gunsalus I.C. Sligar S.G. J. Biol. Chem. 1986; 261: 1158-1163Abstract Full Text PDF PubMed Google Scholar, 4Koga H. Yamaguchi E. Matsunaga K. Aramaki H. Horiuchi T. J. Biochem. ( Tokyo ). 1989; 106: 831-836Crossref PubMed Scopus (74) Google Scholar, 5Peterson J.A. Lorence M.C. Amarneh B. J. Biol. Chem. 1990; 265: 6066-6073Abstract Full Text PDF PubMed Google Scholar). Structural information is available for two of the three subunits of the CYP101 system. CYP101 has been crystallized in a number of states, and the structure is well defined (6Poulos T.L. Finzel B.C. Gunsalus I.C. Wagner G.C. Kraut J. J. Biol. Chem. 1985; 260: 16122-16130Abstract Full Text PDF PubMed Google Scholar, 7Poulos T.L. Finzel B.C. Howard A.J. Biochemistry. 1986; 25: 5314-5322Crossref PubMed Scopus (558) Google Scholar, 8Poulos T.L. Finzel B.C. Howard A.J. J. Mol. Biol. 1987; 195: 687-700Crossref PubMed Scopus (1296) Google Scholar). Structural information on Pdx comes from solution 1H NMR studies by Pochapsky and co-workers (9Ye X.M. Pochapsky T.C. Pochapsky S.S. Biochemistry. 1992; 31: 1961-1968Crossref PubMed Scopus (37) Google Scholar, 10Pochapsky T.C. Ye X.M. Ratnaswamy G. Lyons T.A. Biochemistry. 1994; 33: 6424-6432Crossref PubMed Scopus (110) Google Scholar, 11Pochapsky T.C. Ratnaswamy G. Patera A. Biochemistry. 1994; 33: 6433-6441Crossref PubMed Scopus (39) Google Scholar), and they have proposed a model. Electron transfer in this system proceeds from NADH via the flavin group of PdR to the 2Fe-2S center of Pdx and then to the heme iron of CYP101 which accepts one electron at a time from Pdx. Because the details of the electron transfer pathway from one subunit to the next are missing, it is not known exactly how the subunits bind for the transfer of electrons. Ionic strength is well known to have an effect on binding and electron transfer suggesting that salt bridges are important in these interactions (12Hintz M.J. Peterson J.A. J. Biol. Chem. 1981; 256: 6721-6728Abstract Full Text PDF PubMed Google Scholar, 13Roome Jr., P.W. Philley J.C. Peterson J.A. J. Biol. Chem. 1983; 258: 2593-2598Abstract Full Text PDF PubMed Google Scholar). The role of some amino acid residues, specifically Trp-106 on Pdx and Arg-112 on CYP101, is known to be important for binding and electron transfer (14Davies M.D. Qin L. Beck J.L. Suslick K.S. Koga H. Horiuchi T. Sligar S.G. J. Am. Chem. Soc. 1990; 112: 7396-7398Crossref Scopus (52) Google Scholar, 15Davies M.D. Sligar S.G. Biochemistry. 1992; 31: 11383-11389Crossref PubMed Scopus (65) Google Scholar, 16Koga H. Sagara Y. Yaoi T. Tsujimura M. Nakamura K. Sekimizu K. Makino R. Shimada H. Ishimura Y. Yura K. Go M. Ikeguchi M. Horiuchi T. FEBS Lett. 1993; 331: 109-113Crossref PubMed Scopus (61) Google Scholar, 17Nakamura K. Horiuchi T. Yasukochi T. Sekimizu K. Hara T. Sagara Y. Biochim. Biophys. Acta. 1994; 1207: 40-48Crossref PubMed Scopus (39) Google Scholar, 18Unno 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). Residues involved in the PdR-Pdx interaction are not necessarily the same as those in the Pdx·CYP101 complex. Site-directed mutagenesis of amino acid residues on adrenodoxin, a mitochondrial analog to the camphor hydroxylase, has implicated overlapping but not identical sites for binding of adrenodoxin to its redox partners in the P450ssc system (19Coghlan V.M. Vickery L.E. J. Biol. Chem. 1991; 266: 18606-18612Abstract Full Text PDF PubMed Google Scholar, 20Wada A. Waterman M.R. J. Biol. Chem. 1992; 267: 22877-22882Abstract Full Text PDF PubMed Google Scholar, 21Beckert V. Dettmer R. Bernhardt R. J. Biol. Chem. 1994; 269: 2568-2573Abstract Full Text PDF PubMed Google Scholar). Recently, Sibbesen et al. (22Sibbesen O. De Voss J.J. Ortiz de Montellano P.R. J. Biol. Chem. 1996; 271: 22462-22469Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 23Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual.2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar) developed a functional biocatalyst from a multienzyme system by preparing fusion proteins of the CYP101 system. They linked the three genes in two different sequence orders (PdR-CYP101-Pd and PdR-Pdx-CYP101). Their results showed that the system was not optimal with respect to the rate of product formation. They noted that details of subunit interaction were still obscure. Information on surface interaction sites will lead to further optimization of such self-sufficient systems. In this study we have used site-directed mutagenesis to alter surface residues of Pdx and tested the effect of these mutations in the reconstituted complete CYP101 system as well as on interactions of Pdx with specific redox partners. The purpose is to identify residues that are important for binding and electron transfer and to probe the degree of overlap of the binding sites of PdR-Pdx and Pdx-CYP101. The three protein subunits of the P450cam system were obtained by heterologous expression of the genes in separateEscherichia coli (DH5α) clones. The clones were kindly provided by Dr. J. A. Peterson (University of Texas Southwest Medical Center, Dallas, TX). The gene for Pdx along with some 5′- and 3′-flanking DNA was cloned between a HindIII andSmaI site in the polycloning region of pIBI25 (5Peterson J.A. Lorence M.C. Amarneh B. J. Biol. Chem. 1990; 265: 6066-6073Abstract Full Text PDF PubMed Google Scholar). Mutants of the Pdx gene were obtained by a site-directed mutagenesis protocol (Transformer™, CLONTECH, Palo Alto, CA). 2Certain commercial instruments, reagents, or materials are identified in this paper to specify adequately the experimental procedures. Such identification does not imply recommendation by the National Institute of Standards and Technology, nor does it imply that the materials are necessarily the best available for the purpose. Mutations in the Pdx gene were identified by sequencing both strands of the gene. Sequencing reactions were performed using dye-linked deoxy terminator chemistry (Prism™ Dye-Deoxy Terminator kit, Perkin-Elmer) in a thermocycler (Perkin-Elmer model 9600). The products of the reaction were separated by electrophoresis and analyzed using a DNA sequencer (ABI model 373A DNA Sequencer, Perkin-Elmer). Individual clones, expressing PdR, CYP101, plus wild type and mutant Pdx, were grown in batch culture on undefined rich medium. Protein was released into the supernatant by lysis of the harvested cells, purified in two to three chromatographic steps, and the purity of every batch was monitored spectrophotometrically. The purity of some mutants were confirmed with two-dimensional gel electrophoresis. The details of the above procedures have been recently described (24Grayson D.A. Tewari Y.B. Mayhew M.P. Vilker V.L. Goldberg R.N. Arch. Biochem. Biophys. 1996; 332: 239-247Crossref PubMed Scopus (30) Google Scholar). Purified protein subunits were stored at −80 °C. Protein preparations were concentrated using centrifugal membrane concentrators (Amicon Inc., Beverly, MA; Millipore Corp., Bedford, MA), and the buffer was exchanged using a Sephadex G-10 (Pharmacia Biotech Inc.) column prior to analysis. Mass spectra were recorded on a Finnigan MAT (San Jose, CA) TSQ-70 triple quadrupole mass spectrometer upgraded with a TSQ-700 operating system and equipped with an Analytica (Branford, CT) electrospray source. In the positive ion mode, mass spectra were obtained by scanning the first quadrupole from 1000 to 2000m/z in 2 s at a sampling rate of five data points per mass unit. The charge state ion distribution observed in the electrospray mass spectra were deconvoluted using Finnigan's BIOMASS software. Samples were introduced into the electrospray source by capillary reversed-phase high performance liquid chromatography using a 0.32- × 1200-mm column packed with Vydac C8, 30-nm pore size (The Separations Group, Hesperia, CA). The capillary column was slurry-packed with the aid of a stainless steel pressure vessel (25Moseley M.A. Deterding L.J. Tomer K.B. Jorgenson J.W. Anal. Chem. 1991; 63: 1467-1473Crossref PubMed Scopus (94) Google Scholar) pressurized to 8300 kilopascal with helium. An Ultra-Plus binary gradient micro-LC pump (Microtech Scientific, Sunnyvale, CA) was used to produce the solvent gradients needed for the capillary reversed-phase high performance liquid chromatography. The pump was operated at 40 μl/min, but the column flow rate of 1–2 μl/min was obtained by pre-injector splitting. A Rheodyne (Cotati, CA) model 7520 injector with a 1-μl rotor was used. Prior to LCMS analysis, desalted Pdx samples were diluted to approximately 0.2 g/liter with water. The complete camphor monooxygenase reaction was conducted with all three subunits. The reaction contained Pdx (5 μmol/liter), PdR (0.5 μmol/liter), CYP101 (0.5 μmol/liter), NADH (330 μmol/liter), camphor (550 μmol/liter), KCl (100 mmol/liter) in potassium phosphate buffer (20 mmol/liter), pH 7.4. All components were prepared as stock solutions in the phosphate/KCl buffer. The reaction was started with the addition of NADH. The oxidation of NADH was monitored at 340 nm, and the oxidation of NADH was based on a molar absorptivity of 6.22 (mmol/liter)−1 cm −1. Kinetic studies were done with the complete system keeping the Pdx concentration constant at 5 μmol/liter. The concentration of the redox partner under investigation was varied, and the other partner was present at 0.5 μmol/liter. The kinetic data was analyzed using nonlinear regression analysis (Sigmaplot, Jandel Scientific, San Rafael, CA). Two partial reaction assays, 1) the reduction of Pdx by PdR and 2) the oxidation of reduced Pdx by CYP101, were also performed. These were based on the absorbance difference between oxidized and reduced Pdx at 455 nm (molar absorptivity at 455 nm of reduced Pdx, ε = 2.6 mmol/liter−1 cm−1; and of oxidized Pdx, ε = 10.4 mmol/liter−1 cm−1). A large amount of Pdx was combined with a catalytic amount of PdR or CYP101. This allowed the reaction to proceed at a linear rate measurable over 0.2 min to 0.6 min. Because of the sensitivity of the reaction to small differences in the quantities of redox partners (PdR or CYP101), the results with mutant Pdx proteins were always compared with assays done with the WT Pdx performed on the same day and with the same preparations of other reaction components. The reaction mixture for Pdx reduction consisted of Pdx (100 μmol/liter), NADH (100 μmol/liter), and PdR (10 nmol/liter) in the phosphate/KCl buffer. The reaction was started with the addition of NADH. Absorbance changes were monitored at 455 nm. In the assay monitoring Pdx oxidation, it was necessary to reduce Pdx prior to adding CYP101 and camphor. This was accomplished by using the same reaction mixture that was used for the Pdx reduction assay, raising the concentration of PdR to 20 nmol/liter. After an initial decrease in absorbance of Pdx was observed, resulting from the reduction of Pdx, a steady state absorbance reading was maintained for 1 to 2 min until NADH had been depleted. Then reoxidation of Pdx was observed by linear rate of increase in Pdx absorbance. The rate of nonenzymatic reoxidation remained linear over 0.5 to 1 min and was the same for mutants and WT Pdx, except for W106E (1.5 × WT), and for D38N (2 × WT). Once the pattern and rate of nonenzymatic reoxidation was established for WT and mutant Pdx, assays were started and observed to the point where NADH had been depleted. Then CYP101 (10 nmol/liter) and camphor (200 μmol/liter) were added, and the linear portion of the resulting PdX reoxidation was measured. All values for total reoxidation were corrected for nonenzymatic reoxidation to determine the enzymatic rate of PdX reoxidation. A ribbon model of the structure of Pdx, as proposed by Pochapskyet al. (10Pochapsky T.C. Ye X.M. Ratnaswamy G. Lyons T.A. Biochemistry. 1994; 33: 6424-6432Crossref PubMed Scopus (110) Google Scholar) and based on NMR studies, is shown in Fig. 1. The side chains of the residues mutated in this study are also indicated. The 2Fe-2S cluster, at the middle portion of the molecule as depicted, has four cysteine ligands as follows: Cys-39, Cys-45, Cys-48, and Cys-86 (26Gerber N.C. Horiuchi T. Koga H. Sligar S.G. Biochem. Biophys. Res. Commun. 1990; 169: 1016-1020Crossref PubMed Scopus (24) Google Scholar). The first three lie on a loop leading from the helix on the right (amino acid residues 23–31). The fourth cysteine ligand (Cys-86) is present on another strand coming off a helix, which ends at Cys-73, on the left hand side of the molecule. This helix (residues 65–73) has several acidic residues that were shown in chemical modification studies to be involved in the binding of Pdx to the reductase (27Geren L. Tuls J. O'Brien P. Millett F. Peterson J.A. J. Biol. Chem. 1986; 261: 15491-15495Abstract Full Text PDF PubMed Google Scholar). We chose to mutagenize Cys-73 at the end of this helix. The loop containing three of the four cysteine ligands for the 2Fe-2S center has two acidic residues, Asp-34 and Asp-38. These would be candidates for electrostatic interaction with the series of basic residues in the vicinity of the heme group of CYP101. Recently, Pochapsky et al. (28Pochapsky T.A. Lyons T.A. Kazanis S. Arakaki T. Rasnaswamy G. Biochimie ( Paris ). 1996; 78: 723-733Crossref PubMed Scopus (106) Google Scholar) proposed that these two aspartates and the C-terminal carboxylate of Trp-106 interact with three arginines (Arg-72, Arg-109, and Arg-112) of CYP101. In our studies the acidic residues at positions 34 and 38 were replaced with the amide forms, which neutralized the charge, but did not change the overall size of the side chain. A second substitution of a nonpolar residue was done at position 38 (D38I), and cysteine was substituted for serine at position 42 which is also in the region near the Fe-S center of Pdx. Trp-106 has already been shown to have an important role in the Pdx·CYP101 complex with the most important aspect involving the aromatic nature of the amino acid (14Davies M.D. Qin L. Beck J.L. Suslick K.S. Koga H. Horiuchi T. Sligar S.G. J. Am. Chem. Soc. 1990; 112: 7396-7398Crossref Scopus (52) Google Scholar,15Davies M.D. Sligar S.G. Biochemistry. 1992; 31: 11383-11389Crossref PubMed Scopus (65) Google Scholar). The mutant W106E is similar to one (W106D) previously studied by Davies and co-workers (14Davies M.D. Qin L. Beck J.L. Suslick K.S. Koga H. Horiuchi T. Sligar S.G. J. Am. Chem. Soc. 1990; 112: 7396-7398Crossref Scopus (52) Google Scholar, 15Davies M.D. Sligar S.G. Biochemistry. 1992; 31: 11383-11389Crossref PubMed Scopus (65) Google Scholar). In addition, three residues were chosen to test the hypothesis that they lie outside the interaction surface of Pdx with its redox partners and therefore could serve as controls: the charge was neutralized on His-8 by replacement with a tyrosine; the charge was reversed on Asp-95 by replacement with a histidine; and the size of the side chain at Ala-55 was increased by replacement with a valine. Mutant Pdx species were produced by a site-directed mutagenesis protocol. The identity of all of the mutations were verified two ways: 1) both strands of the mutated gene were sequenced and 2) the molecular weight of each mutant Pdx protein species was determined by electrospray mass spectrometry (Table I). The mutated Pdx proteins were expressed in E. coli (DH5α) cells on the plasmid PIBI25 as was used for expression of the WT. The level of expression of the mutants in E. coli varied from the wild type. In most cases, the yield of protein (after purification) was somewhat lower than WT Pdx (3.6 mg of pure protein/g of cell FW). However, C73R expressed at higher levels than WT. The mutant proteins were purified by the same protocol as used for the WT, and purity was monitored spectroscopically according to Gunsalus and Wagner (29Gunsalus I.C. Wagner G.C. Methods Enzymol. 1978; 52: 166-188Crossref PubMed Scopus (302) Google Scholar). Wavelength scans of the mutants were identical to the WT with the exceptions noted below. Based on the ratio of absorbances at 455 and 275 nm, the mole fraction purity of WT and mutants was greater than 0.99. The purity of two mutants, W106E and H8Y, was verified by two-dimensional gel electrophoresis because the absorbance at 275 nm was altered by the mutation, invalidating the 455/275 ratio. Although no specific study of the stability of the mutant protein species was made, there was no detectable degeneration of the mutant or WT Pdx protein over the period of hours when assays were conducted.Table IMolecular mass of native and mutant putidaredoxin species determined by electrospray mass spectrometryPutidaredoxinMr(calculated)Mr (measured)Wild type11,419.011,418 ± 2C73S11,402.911,404 ± 2C73G11,372.911,371 ± 2C73R11,472.111,471 ± 3S42C11,435.111,434 ± 4D34N11,418.011,416 ± 2D38N11,418.011,416 ± 2D38I11,417.111,414 ± 4W106E11,361.911,360 ± 2D95H11,441.111,442 ± 2H8Y11,445.011,442 ± 2A55V11,447.111,447 ± 2The calculated and observed values of Mr are for the apoprotein of the Pdx species. Numbers for the observed values are ±95% confidence limits of the average observed Mr. Open table in a new tab The calculated and observed values of Mr are for the apoprotein of the Pdx species. Numbers for the observed values are ±95% confidence limits of the average observed Mr. The complete camphor P450 hydroxylase system was reconstituted, and the activity of the mutant Pdx species, measured as camphor-dependent NADH oxidation, was compared with that of the WT protein (Table II). All three substitutions made at Cys-73 proved to have a significant effect on activity. The substitution of serine for cysteine resulted in loss of half of the activity. The glycine and arginine mutants at Cys-73 proved even more disruptive, producing 36 and 5% WT Pdx activity, respectively. In contrast to the substitution of serine for cysteine at position 73 (C73S), the opposite mutation at position 42 (S42C) was less detrimental (64% WT). The two substitutions at Asp-38 were dramatic in their effects, with the substitution of isoleucine resulting in almost complete loss of activity (<1%), whereas the D38N mutant had 10% WT activity, much less than the D34N mutant (70% WT). The result seen with the W106E substitution was the same (3%) as observed by Davies et al. (14Davies M.D. Qin L. Beck J.L. Suslick K.S. Koga H. Horiuchi T. Sligar S.G. J. Am. Chem. Soc. 1990; 112: 7396-7398Crossref Scopus (52) Google Scholar) with a W106A mutant (2% WT activity). The remaining mutations, D95H, H8Y, and A55V, had no effect on activity, suggesting that these residues are likely to be outside of the interaction sites, and the change in charge of two of these was of no consequence.Table IINADH oxidation by the complete P-450cam system in the presence of camphorMutant putidaredoxinWild type putidaredoxin, NADH oxidationIdentityNADH oxidationMutant Pdx as a % of WT activitys −1%s −1C73S5.6 (0.4)4213.2 (1.1)C73G4.7 (0.6)3613.2 (0.4)C73R0.7 (0.03)613.3 (1.3)S42C8.8 (1.6)6413.8 (1.8)D34N9.1 (0.3)7013.1 (0.3)D38N1.1 (0.1)814.1 (0.2)D38I0.04 (0.02)<114.7 (0.6)W106E0.4 (0.04)314.1 (0.4)D95H14.6 (0.6)10114.5 (0.6)H8Y14.0 (0.6)10213.7 (0.4)A55V14.2 (0.8)9814.5 (0.8)Overall average13.9 (0.9)Multiple assays of each mutant Pdx were conducted on at least two separate occasions according to the protocol outlined under “Experimental Procedures.” Assays with WT Pdx were also conducted on the same day with the same preparations of proteins and substrates as were used to determine the activity of the mutant Pdx. Activity of a mutant, as a percent of WT activity, is based on the average WT Pdx activity determined at the same time as the mutant. Numbers in parentheses are 1 S.D. Open table in a new tab Multiple assays of each mutant Pdx were conducted on at least two separate occasions according to the protocol outlined under “Experimental Procedures.” Assays with WT Pdx were also conducted on the same day with the same preparations of proteins and substrates as were used to determine the activity of the mutant Pdx. Activity of a mutant, as a percent of WT activity, is based on the average WT Pdx activity determined at the same time as the mutant. Numbers in parentheses are 1 S.D. Partial or half assays were performed to explore whether the loss of activity by the mutant Pdx proteins was the result of defective interactions with one or both of the redox partners. To do these measurements, we took advantage of the absorbance difference in Pdx at 455 nm as a function of redox state (Fig. 2). We separately measured 1) rates of Pdx reduction by PdR and NADH (Table III) and 2) rates of camphor and CYP101-dependent oxidation of reduced Pdx (Table IV).Table IIIPutidaredoxin reduction by putidaredoxin reductase and NADHMutant putidaredoxinWild type putidaredoxin, Pdx reducedIdentityPdx reducedMutant Pdx activity as a % of WT activitynmol min −1%nmol min −1C73S23.3 (2.1)7730.2 (0.7)C73G17.4 (1.8)6029.2 (0.5)C73R4.3 (0.2)1527.7 (2.1)S42C31.2 (1.7)10031.1 (1.1)D34N30.6 (1.1)11227.3 (1.4)D38N33.0 (4.0)12126.4 (2.4)D38I7.9 (0.3)2729.1 (2.0)W106E18.1 (1.1)6727.1 (2.8)D95H29.7 (0.7)10029.8 (1.1)H8Y32.4 (1.2)10431.2 (1.0)A55V28.3 (2.2)10127.9 (2.4)Overall average28.5 (2.5)See legend for Table II. Open table in a new tab Table IVOxidation of reduced putidaredoxin by P450 and camphorMutant putidaredoxinWild type putidaredoxin, Pdx oxidizedIdentityPdx oxidizedMutant Pdx activity as a % of WT activitynmol min −1%nmol min −1C73S16.0 (2.9)9117.6 (1.5)C73G16.9 (3.0)9517.8 (2.2)C73R14.3 (2.0)9215.5 (3.5)S42C11.2 (1.9)6916.2 (1.2)D34N13.1 (1.1)9314.1 (1.7)D38N3.4 (0.3)2215.3 (2.1)D38I1.1 (0.6)616.6 (1.6)W106E1.7 (0.9)1115.6 (1.0)D95H15.6 (2.2)9316.7 (3.2)H8Y14.9 (2.1)9815.2 (2.5)A55V16.5 (1.1)10316.0 (1.1)Overall average16.3 (2.3)See legend for Table II. Open table in a new tab See legend for Table II. See legend for Table II. Overall, the pattern of activity differences of the mutant Pdx proteins with respect to WT Pdx that was observed in the half-assay reactions was consistent with the differences seen in the complete assay. The three mutant Pdx proteins (D95H, H8Y, and A55V), which showed no changes in the complete camphor-dependent NADH oxidation reaction (Table II), were either the same or not statistically different from the wild type values for the partial reactions. The effect of the mutations at Cys-73 was seen in the interaction of Pdx with PdR but not with CYP101 (Tables III and IV). The primary effect of the mutations at Asp-38, Ser-42, and Trp-106 was on the interaction of Pdx with the CYP101. The W106E mutant exhibited a low rate of activity with CYP101 as would be expected from other studies where substitution of tryptophan with anything other than another aromatic residue reduced activity to very low levels (14Davies M.D. Qin L. Beck J.L. Suslick K.S. Koga H. Horiuchi T. Sligar S.G. J. Am. Chem. Soc. 1990; 112: 7396-7398Crossref Scopus (52) Google Scholar, 15Davies M.D. Sligar S.G. Biochemistry. 1992; 31: 11383-11389Crossref PubMed Scopus (65) Google Scholar). The substitution of asparagine for aspartic acid at residue 38 had a significant negative effect on the interaction with CYP101 reducing the activity to 22% WT. Substituting the non-polar residue isoleucine for Asp-38 was even more detrimental, nearly eliminating activity with CYP101. The S42C mutation clearly affected the interaction of Pdx with CYP101, whereas that with PdR was 100% WT. The results of the partial assay measurements with the D34N Pdx mutant presented an anomaly in that there appeared to be a small effect on the interaction of reduced D34N Pdx with CYP101, whereas the activity of this mutant in the total camphor reaction was 30% lower than WT. The effects of the mutation of residues Asp-38, Asp-34, and Trp-106 were not restricted just to interaction with the CYP101 subunit, which provides some evidence for overlap of binding sites on Pdx for PdR and CYP101. The substitution of asparagine at Asp-34 and Asp-38 appeared to result in a small enhancement of activity with respect to PdR. However, the positive effect disappeared with the substitution of the non-polar isoleucine for Asp-38, which resulted in the loss of activity. W106E had somewhat impaired interaction with PdR (67% WT activity), although not to the same degree as its interaction with CYP101. A Michaelis-Menten kinetic analysis was conducted on some of the mutant Pdx species to investigate further the nature of the changes in the interaction between redox partners. These studies were done with the complete camphor assay, measuring NADH oxidation (Table V and Fig. 3). In kinetic studies the reacting substrates are maintained in nonlimiting quantities except for the substrate under investigation. But in this study, the protein subunits were being treated as substrates. It was not possible, for example, to provide unlimited quantities of CYP101 while investigating the kinetics of the reaction with regard to PdR and still be able to measure a linear rate of NADH oxidation over a reasonable period. Therefore, the kinetic parameter values are somewhat specific to this particular assay reaction. The value of the measurements rests not in the absolute numbers but in the comparison with the WT Pdx values.Table VDetermination of apparent kinetic parameters of redox partners for mutant and wild type putidaredoxinPutidaredoxinPdRP450camKmVmaxKmVmaxμ mNADH Ox. s −1μ mNADH Ox. s −1Wild type0.53 (0.03)31 (0.9)0.33 (0.04)24 (2.0)D95H0.55 (0.01)30 (2.8)0.42 (0.12)27 (2.3)C73S2.26 (0.24)33 (3.6)C73G2.24 (0.25)29 (2.9)C73R9.23 (0.72)18 (0.8)S42C1.33 (0.1)30 (3.7)D38N5.96 (1.9)12 (1.3)D34N0.65 (0.2)22 (3.8)W106E20.1 (4.8)11 (2.8)For these experiments the camphor-dependent NADH oxidation was measured with the complete P450cam monooxygenase system as described under “Experimental Procedures.” The concentration of the redox partner under investigation was varied, whereas the concentration of Pdx and the other redox partner was held constant. The data were analyzed using nonlinear regression analysis. The determination for each kinetic parameter was completed three or more times. The values are avera