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• W2145194877 abstract "Site-directed mutagenesis of Thr66 in porcine liver NADH-cytochromeb 5 reductase demonstrated that this residue modulates the semiquinone form of FAD and the rate-limiting step in the catalytic sequence of electron transfer. The absorption spectrum of the T66V mutant showed a typical neutral blue semiquinone intermediate during turnover in the electron transfer from NADH to ferricyanide but showed an anionic red semiquinone form during anaerobic photoreduction. The apparent k cat values of this mutant were ∼10% of that of the wild type enzyme (WT). These data suggest that the T66V mutation stabilizes the neutral blue semiquinone and that the conversion of the neutral blue to the anionic red semiquinone form is the rate-limiting step. In the WT, the value of the rate constant of FAD reduction (k red) was consistent with thek cat values, and the oxidized enzyme-NADH complex was observed during the turnover with ferricyanide. This indicates that the reduction of FAD by NADH in the WT-NADH complex is the rate-limiting step. In the T66A mutant, thek red value was larger than thek cat values, but thek red value in the presence of NAD+was consistent with the k cat values. The spectral shape of this mutant observed during turnover was similar to that during the reduction with NADH in the presence of NAD+. These data suggest that the oxidized T66A-NADH-NAD+ ternary complex is a major intermediate in the turnover and that the release of NAD+ from this complex is the rate-limiting step. These results substantiate the important role of Thr66 in the one-electron transfer reaction catalyzed by this enzyme. On the basis of these data, we present a new kinetic scheme to explain the mechanism of electron transfer from NADH to one-electron acceptors including cytochromeb 5. Site-directed mutagenesis of Thr66 in porcine liver NADH-cytochromeb 5 reductase demonstrated that this residue modulates the semiquinone form of FAD and the rate-limiting step in the catalytic sequence of electron transfer. The absorption spectrum of the T66V mutant showed a typical neutral blue semiquinone intermediate during turnover in the electron transfer from NADH to ferricyanide but showed an anionic red semiquinone form during anaerobic photoreduction. The apparent k cat values of this mutant were ∼10% of that of the wild type enzyme (WT). These data suggest that the T66V mutation stabilizes the neutral blue semiquinone and that the conversion of the neutral blue to the anionic red semiquinone form is the rate-limiting step. In the WT, the value of the rate constant of FAD reduction (k red) was consistent with thek cat values, and the oxidized enzyme-NADH complex was observed during the turnover with ferricyanide. This indicates that the reduction of FAD by NADH in the WT-NADH complex is the rate-limiting step. In the T66A mutant, thek red value was larger than thek cat values, but thek red value in the presence of NAD+was consistent with the k cat values. The spectral shape of this mutant observed during turnover was similar to that during the reduction with NADH in the presence of NAD+. These data suggest that the oxidized T66A-NADH-NAD+ ternary complex is a major intermediate in the turnover and that the release of NAD+ from this complex is the rate-limiting step. These results substantiate the important role of Thr66 in the one-electron transfer reaction catalyzed by this enzyme. On the basis of these data, we present a new kinetic scheme to explain the mechanism of electron transfer from NADH to one-electron acceptors including cytochromeb 5. cytochromeb 5 solubilized domain of porcine liver cytochrome b 5 NADH-cytochromeb 5 reductase solubilized catalytic domain of the porcine liver NADH-cytochrome b 5reductase ferredoxin-NADP+ oxidoreductase wild type recombinant Pb5R NADH-cytochrome b 5 reductase (EC 1.6.2.2) is a member of the large family of flavin-dependent oxidoreductases that transfer an electron from two-electron carriers of nicotinamide dinucleotides to one-electron carriers such as heme proteins and ferredoxins. This enzyme catalyzes the electron transfer from NADH to cytochrome b 5(b5)1 (1Spatz L. Strittmatter P. J. Biol. Chem. 1973; 248: 793-799Abstract Full Text PDF PubMed Google Scholar, 2Okayasu T. Nagao M. Ishibashi T. Imai Y. Arch. Biochem. Biophys. 1981; 206: 21-28Crossref PubMed Scopus (109) Google Scholar, 3Iyanagi T. Watanabe S. Anan K.F. Biochemistry. 1984; 23: 1418-1425Crossref PubMed Scopus (70) Google Scholar), and participates in fatty acid synthesis (4Oshino N. Imai Y. Sato R. J. Biochem. (Tokyo). 1971; 69: 155-167Crossref PubMed Scopus (316) Google Scholar, 5Keyes S.R. Cinti D.L. J. Biol. Chem. 1980; 255: 11357-11364Abstract Full Text PDF PubMed Google Scholar), cholesterol synthesis (6Reddy V.V. Kupfer D. Capsi E. J. Biol. Chem. 1977; 252: 2797-2801Abstract Full Text PDF PubMed Google Scholar), and xenobiotic oxidation (7Hildebrandt A. Estabrook R.W. Arch. Biochem. Biophys. 1971; 143: 66-79Crossref PubMed Scopus (454) Google Scholar) as a member of the electron transport chain on the endoplasmic reticulum. In erythrocytes, this enzyme participates in the reduction of methemoglobin (8Hultquist D.E. Passon P.G. Nat. New Biol. 1971; 229: 252-254Crossref PubMed Scopus (256) Google Scholar). The outline of the catalytic cycle of the solubilized catalytic domain of NADH-cytochrome b 5 reductase (b5R) is understood as follows (3Iyanagi T. Watanabe S. Anan K.F. Biochemistry. 1984; 23: 1418-1425Crossref PubMed Scopus (70) Google Scholar) (Scheme FSI). At first, two electrons are transferred from NADH to FAD by hydride (H−) transfer. Then the two-electron reduced enzyme-NAD+ complex (E-FADH−-NAD+) transfers two electrons to two one-electron acceptors one by one via the anionic red semiquinone form (E-FAD·−-NAD+), and the reduced enzyme returns to the oxidized state. Strittmatter (9Strittmatter P. J. Biol. Chem. 1962; 237: 3250-3254Abstract Full Text PDF PubMed Google Scholar, 10Strittmatter P. J. Biol. Chem. 1963; 238: 2213-2219Abstract Full Text PDF PubMed Google Scholar, 11Strittmatter P. J. Biol. Chem. 1965; 240: 4481-4487Abstract Full Text PDF PubMed Google Scholar) suggested that the reduction of FAD by NADH is the rate-limiting step in electron transfer catalyzed by b5R. Iyanagi et al. (3Iyanagi T. Watanabe S. Anan K.F. Biochemistry. 1984; 23: 1418-1425Crossref PubMed Scopus (70) Google Scholar, 12Iyanagi T. Biochemistry. 1977; 16: 2725-2730Crossref PubMed Scopus (83) Google Scholar) found that the anionic red semiquinone of FAD in Pb5R is stabilized by binding of NAD+. Kobayashi et al. (13Kobayashi K. Iyanagi T. Ohara H. Hayashi K. J. Biol. Chem. 1988; 263: 7493-7499Abstract Full Text PDF PubMed Google Scholar) analyzed the conversion of the neutral blue to the red semiquinone in the presence of NAD+ using a pulse radiolysis technique. Meyeret al. (14Meyer T.E. Shirabe T. Yubisui M. Takeshita M. Bes M.T. Cusanovich M.A. Tollin G. Arch. Biochem. Biophys. 1995; 318: 457-469Crossref PubMed Scopus (16) Google Scholar) also demonstrated that NAD+stabilizes the red semiquinone of the human b5R and modulates the electron transfer to b5. These studies suggest the importance of the anionic red semiquinone form of the b5R-NAD+ complex in the electron transfer. The preliminary tertiary structure of human erythrocyte b5R (15Takano T. Ogawa K. Sato M. Bando S. Yubisui T. J. Mol. Biol. 1987; 195: 749-750Crossref PubMed Scopus (8) Google Scholar, 16Takano T. Bando S. Horii C. Higashiyama M. Ogawa K. Sato M. Katsuya Y. Dannno M. Yubisui T. Shirabe K. Takeshita M. Yagi K. Flavins and Flavoproteins. Walter de Gruyter, Berlin1994: 409-412Google Scholar), and the detailed tertiary structures of porcine and rat liver b5Rs at 2.1 Å resolution have been determined by x-ray crystallography (17Miki K. Kaida S. Kasai N. Iyanagi T. Kobayashi K. Hayashi K. J. Biol. Chem. 1987; 262: 11801-11802Abstract Full Text PDF PubMed Google Scholar, 18Nishida H. Inaka K. Yamanaka M. Kaida S. Kobayashi K. Miki K. Biochemistry. 1995; 34: 2763-2767Crossref PubMed Scopus (88) Google Scholar, 19Nishida H. Inaka K. Miki K. FEBS Lett. 1995; 361: 97-100Crossref PubMed Scopus (24) Google Scholar, 20Nishida H. Miki K. Proteins. 1996; 26: 32-41Crossref PubMed Scopus (27) Google Scholar, 21Bewley M.C. Marohnic C.C. Barber M.J. Biochemistry. 2001; 40: 13574-13582Crossref PubMed Scopus (72) Google Scholar). These structural studies revealed that NADH-cytochromeb 5 reductase belongs to the structurally related so-called “ferredoxin reductase family” (22Karplus P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-65Crossref PubMed Scopus (464) Google Scholar, 23Correll C.C. Ludwig M.L. Bruns C.M. Karplus P.A. Protein Sci. 1993; 2: 2112-2133Crossref PubMed Scopus (162) Google Scholar) together with other flavoenzymes such as ferredoxin-NADP+ oxidoreductase (FNR) (22Karplus P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-65Crossref PubMed Scopus (464) Google Scholar), phthalate dioxygenase reductase (24Correll C.C. Batie C.J. Ballow D.P. Ludwig M.L. Science. 1992; 258: 1604-1610Crossref PubMed Scopus (264) Google Scholar), flavodoxin reductase (25Ingelman M. Bianchi V. Eklund H. J. Mol. Biol. 1997; 268: 147-157Crossref PubMed Scopus (124) Google Scholar), NADPH-cytochrome P-450 reductase (26Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (671) Google Scholar), and the cytochromeb reductase domain of nitrate reductase (27Lu G. Campbell W.H. Schneider G. Lindqvist Y. Structure. 1994; 2: 809-821Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Enzymes of this family contain a flavin-binding domain and a pyridine nucleotide-binding domain. The former domain has a highly conserved flavin-binding amino acid sequence motif, RXY(T/S). In the Pb5R, Arg63, Tyr65, and Thr66comprise this sequence motif (19Nishida H. Inaka K. Miki K. FEBS Lett. 1995; 361: 97-100Crossref PubMed Scopus (24) Google Scholar). Using site-directed mutagenesis, we demonstrated that the positive charge of Arg63 is critical for the affinities of Pb5R for both NADH and NAD+, and the specific arrangement between the side chain of Tyr65 and FAD contributes to protein stability and electron transfer (28Kimura S. Nishida H. Iyanagi T. J. Biochem. (Tokyo). 2001; 130: 481-490Crossref PubMed Scopus (25) Google Scholar). Marohnic and Barber (29Marohnic C.C. Barber M.J. Arch. Biochem. Biophys. 2001; 389: 223-233Crossref PubMed Scopus (27) Google Scholar) also reported the effects of mutations of the corresponding Arg91 in rat b5R. The Thr66 residue in Pb5R is positioned near both the N5 atom of the isoalloxazine ring of FAD and the potential binding site of the nicotinamide ring of NADH (20Nishida H. Miki K. Proteins. 1996; 26: 32-41Crossref PubMed Scopus (27) Google Scholar, 28Kimura S. Nishida H. Iyanagi T. J. Biochem. (Tokyo). 2001; 130: 481-490Crossref PubMed Scopus (25) Google Scholar) (Fig. 1). This position corresponds to threonine or serine residues in the other members of the ferredoxin reductase family (22Karplus P.A. Daniels M.J. Herriott J.R. Science. 1991; 251: 60-65Crossref PubMed Scopus (464) Google Scholar, 24Correll C.C. Batie C.J. Ballow D.P. Ludwig M.L. Science. 1992; 258: 1604-1610Crossref PubMed Scopus (264) Google Scholar, 25Ingelman M. Bianchi V. Eklund H. J. Mol. Biol. 1997; 268: 147-157Crossref PubMed Scopus (124) Google Scholar, 26Wang M. Roberts D.L. Paschke R. Shea T.M. Masters B.S.S. Kim J.J.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8411-8416Crossref PubMed Scopus (671) Google Scholar, 27Lu G. Campbell W.H. Schneider G. Lindqvist Y. Structure. 1994; 2: 809-821Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). Ser96 in spinach leaf FNR is critical to the reductive half-reaction of FAD (30Aliverti A. Bruns C.M. Pandini V.E. Karplus P.A. Vanoni M.A. Curti B. Zanetti G. Biochemistry. 1995; 34: 8371-8379Crossref PubMed Scopus (63) Google Scholar), and Ser90 in the C-terminal Tyr208 mutant of pea leaf FNR forms a hydrogen bond with the amide moiety on the nicotinamide ring of the pyridine nucleotide in both the enzyme-NADP+ and enzyme-NADPH complexes (31Deng Z. Aliverti A. Zanetti G. Arakaki A.K. Ottado J. Orellano E.G. Calcaterra N.B. Ceccarell E.A. Carrillo N. Karplus P.A. Nat. Struct. Biol. 1999; 6: 847-853Crossref PubMed Scopus (184) Google Scholar). However, b5R and cytochrome b reductase domain of nitrate reductase do not have an aromatic ring corresponding to that of the C-terminal Tyr208 in pea leaf FNR, which contacts with there-side of the isoalloxazine ring of FAD and moves away accompanied with the binding of nicotinamide (31Deng Z. Aliverti A. Zanetti G. Arakaki A.K. Ottado J. Orellano E.G. Calcaterra N.B. Ceccarell E.A. Carrillo N. Karplus P.A. Nat. Struct. Biol. 1999; 6: 847-853Crossref PubMed Scopus (184) Google Scholar). In addition, the main physiological role of leaf FNR is the reduction of the oxidized pyridine nucleotide, and the direction of the electron transfer between FAD and pyridine nucleotide is different from that of b5R. Therefore, it is considered that Thr66 in Pb5R contributes to the reduction of FAD and/or the stabilization of the reduced FAD. Shirabeet al. (32Shirabe K. Yubisui T. Takeshita M. Yagi K. Flavins and Flavoproteins. Walter de Gruyter, Berlin1994: 405-408Google Scholar) reported that mutations of the corresponding Thr94 in human b5R affect oxidation of FAD, but the effects of the mutations on the properties of reduced FAD in the catalytic cycle have not been clarified. To analyze the role of Thr66 in catalysis in Pb5R, we replaced Thr66 in Pb5R with serine (T66S), alanine (T66A), and valine (T66V) and analyzed the redox properties of FAD in the catalytic cycle using a stopped flow spectrophotometer. We present here that the conversion of the neutral blue to the red semiquinone intermediate and the release of NAD+ from the enzyme in the catalytic cycle were modulated by the mutations of Thr66 in Pb5R. In addition, we present a new model of the reaction sequence of Pb5R containing the blue neutral semiquinone and the oxidized enzyme-NAD+-NADH ternary complex. Enzymes for recombinant DNA technology were from Takara and Toyobo. NADH and NAD+ were from Oriental Yeast. Wild type recombinant Pb5R (WT) was prepared as previously described (33Kimura S. Emi Y. Ikushiro S. Iyanagi T. Biochim. Biophys. Acta. 1999; 1430: 290-301Crossref PubMed Scopus (14) Google Scholar). Alteration of the gene encoding Pb5R was carried out by site-directed mutagenesis using PCR by the methods described by Higuchi (34Higuchi R. Innis M.A. Gelfand D.H. Shinsky J.J. White T.J. PCR Protocols: A Guide to Methods and Applications. Academic Press, San Diego, CA1990: 177-183Google Scholar). Briefly, for the preparation of the mutant genes encoding the mutant proteins, two primary PCR products that overlap in sequence were first obtained from a DNA template, pU8Pb5R, which contains the gene encoding the WT (33Kimura S. Emi Y. Ikushiro S. Iyanagi T. Biochim. Biophys. Acta. 1999; 1430: 290-301Crossref PubMed Scopus (14) Google Scholar). One product was generated with the forward primer 5′-TAGGAGGTCATATGTCCACCCCGGCC-3′ containing a NdeI site (underlined) and the mutagenic common reverse primer, 5′-GGGCCGAATGACCAG-3′, and the other was obtained with the forward mutagenic primer and the reverse primer 5′-CCGCCAAGCTTCTAGAAGGCGAAGCAGC-3′ containing aHindIII site (underlined). As the mutagenic forward primers, 5′-CTGGTCATTCGGCCCTACNNNCCCGTCTC-3′, which have a complementary nucleotide sequence to the 5′-end of the mutagenic forward primers, were used. In these primers, NNN are the bases corresponding to the 66th amino acid residue, and GCT, TCG, and GTG were used for the mutations to alanine, serine, and valine, respectively. The resultant two PCR products were mixed and reamplified with the forward and reverse primers. The resultant secondary PCR product was inserted into the plasmid pCWori+ (35Barnes H.J. Arlotto M.P. Waterman M.R. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 5597-5601Crossref PubMed Scopus (543) Google Scholar), a derivative of pHSe5 (36Muchmore D.C. McIntosh L.P. Russell C.B. Anderson D.E. Dahlquist F.W. Oppenheimer N.J. Methods in Enzymology. Academic Press, New York1989: 44-73Google Scholar, 37Gegner J.A. Dahlquist F.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 750-754Crossref PubMed Scopus (166) Google Scholar), using the NdeI and HindIII sites to construct the expression plasmid for generating mutant proteins. The entire nucleotide sequences of the mutant genes were confirmed using an ABI PRISM 310 Genetic Analyzer. All of the mutant proteins were expressed in the soluble fraction of Escherichia coli BL21 cells and purified using the same method as the WT (33Kimura S. Emi Y. Ikushiro S. Iyanagi T. Biochim. Biophys. Acta. 1999; 1430: 290-301Crossref PubMed Scopus (14) Google Scholar). The purity of the mutant proteins was confirmed by SDS-PAGE using 12% polyacrylamide gels. The flavin bound to the mutant proteins was analyzed by TLC on a Kieselgel 60 F245 plate (Merck) (38Fazecas A.G. Kokai K. McCormick D.B. Wright L.D. Methods in Enzymology. Academic Press, New York1971: 385-398Google Scholar). Purified mutant proteins were stored in 100 mm potassium phosphate (pH 7.0) containing 0.1 mm EDTA at −20 °C until use. The recombinant solubilized domain of porcine liver cytochrome b 5 (Pb5) was prepared as follows. The cDNA encoding the full-length porcine liver cytochrome b 5 was amplified from the previously described first strand cDNA, which was prepared from a total RNA preparation from porcine liver (33Kimura S. Emi Y. Ikushiro S. Iyanagi T. Biochim. Biophys. Acta. 1999; 1430: 290-301Crossref PubMed Scopus (14) Google Scholar). The forward primer was 5′-GTTAAGAAATGGCCGAGGAGTCC-3′, which has an initiator methionine codon followed by the nucleotide sequence encoding the N-terminal tetrapeptide of natural bovine liver b5 (39Cristiano R.J. Steggles A.W. Nucleic Acids Res. 1989; 17: 799Crossref PubMed Scopus (26) Google Scholar). The reverse primer was 5′-CTTCGGTTACCTTCTTTTCTGACG-3′. This nucleotide sequence was complementary to the nucleotide sequence located 12–35 bases downstream after the stop codon in the cDNA of bovine liver b5 (39Cristiano R.J. Steggles A.W. Nucleic Acids Res. 1989; 17: 799Crossref PubMed Scopus (26) Google Scholar). The amplified DNA fragment was blunt-ended and inserted into theHincII site of plasmid pUC118, and plasmid pU8Pb5 was selected. Plasmid pU8Pb5 contained the nucleotide sequence encoding 133 amino acid residues from the N-terminal Ala1 to the C-terminal Asn133 of porcine liver b5. The deduced amino acid sequence was identical to that previously reported except for the difference at position 3 (40Ozols J. Biochemistry. 1974; 13: 426-434Crossref PubMed Scopus (82) Google Scholar, 41Abe K. Kimura S. Kizawa R. Anan F.K. Sugita Y. J. Biochem. (Tokyo). 1985; 97: 1659-1668Crossref PubMed Scopus (19) Google Scholar, 42Van Der Mark P.K. Steggles A.W. Biochem. Biophys. Res. Commun. 1997; 240: 80-83Crossref PubMed Scopus (13) Google Scholar). The deduced amino acid residue at position 3 was not glutamine but glutamic acid. The polypeptide containing 87 amino acid residues from Ala7 to Lys93 was prepared as recombinant Pb5. The cDNA encoding recombinant Pb5 was amplified from pU8Pb5 with the forward primer 5′-AGGAGGTCATATGGCCGTGAAGTATTACACC-3′, which has anNdeI site (underlined) containing an additional initiator methionine codon, followed by the nucleotide sequence encoding the N-terminal hexapeptide of recombinant Pb5, and the reverse primer 5′-CCGCCAAGCTTCTACTTGGCAATCTTGATC-3′, which corresponds to the C-terminal peptide, a stop codon, and aHindIII site (underlined). The resultant fragment was inserted into pCWori+ using NdeI and HindIII sites to construct pCPb5. E. coli TG1 cells containing pCPb5 were cultivated in Luria-Bertani medium containing 50 μg/ml ampicillin at 37 °C. When the absorbance at 600 nm was ∼0.3, isopropyl-β-d-thiogalactopyranoside was added to a final concentration of 0.2 mm, and the cultivation was continued for 14 h. The cells were lysed by sonication in 10 mm Tris-HCl (pH 7.4) containing 1 mm EDTA and 2 mm phenylmethylsulfonyl fluoride. The lysate was subjected to centrifugation at 18,000 ×g for 20 min, and the supernatant was separated on a Sephadex G-100 (Pharmacia) column equilibrated with 10 mmpotassium phosphate (pH 7.0) (buffer A). Red colored fractions containing recombinant Pb5 were applied to an anion exchange resin (DE52) column equilibrated with buffer A, and the proteins were eluted with a linear gradient of potassium chloride from 0 to 0.4m in buffer A. Fractions containing recombinant Pb5 were concentrated and desalted with a Sephadex G-25 (fine) (Pharmacia) column equilibrated with 100 mm potassium phosphate (pH 7.0). The purified recombinant Pb5 showed a single band of ∼10 kDa on a 15% polyacrylamide gel after SDS-PAGE. The yield of the purified protein from 1 liter of culture fluid was 5.9 mg. The N-terminal amino acid sequence analyzed with a Shimadzu PSQ-1 protein sequencer was Ala-Val-Lys-Tyr-Tyr, and most of the additional N-terminal methionine residues were cleaved. The oxidized and reduced absorption spectra at 300–700 nm and the ability to accept electrons from the Pb5R were almost identical to those of the natural Pb5 (43Omura T. Takesue S. J. Biochem. (Tokyo). 1970; 67: 249-257Crossref PubMed Scopus (656) Google Scholar). The molar extinction coefficients of the mutant proteins at 460 nm (ε460) were determined by a method similar to that described by Aliverti and Zanetti (44Aliverti A. Zanetti G. Biochemistry. 1997; 36: 14771-14777Crossref PubMed Scopus (17) Google Scholar), as previously described (33Kimura S. Emi Y. Ikushiro S. Iyanagi T. Biochim. Biophys. Acta. 1999; 1430: 290-301Crossref PubMed Scopus (14) Google Scholar). The molar concentration of the WT was determined from the absorbance at 460 nm using the molar extinction coefficient, 1.02 × 104 m−1cm−1 (33Kimura S. Emi Y. Ikushiro S. Iyanagi T. Biochim. Biophys. Acta. 1999; 1430: 290-301Crossref PubMed Scopus (14) Google Scholar). The protein concentration of recombinant Pb5 was determined using the molar extinction coefficient of natural Pb5, 1.13 × 105m−1cm−1 at 413 nm (45Strittmatter P. Boyer E.D. Lardy H.A. Mybäck K. The Enzymes. Academic Press, New York1963: 113-145Google Scholar). Absorption spectra were measured on a Hitachi U-2010 spectrophotometer. CD spectra were measured on a Jasco J-700 spectropolarimeter. Fluorescent emission spectra were measured in 10 mm potassium phosphate (pH 7.0) at 25 °C on a Hitachi F-3010 Fluorescence spectrophotometer. The excitation wavelength was 460 nm, and emission spectra at 470–650 nm were observed. Steady-state enzymatic activities were measured as previously described (33Kimura S. Emi Y. Ikushiro S. Iyanagi T. Biochim. Biophys. Acta. 1999; 1430: 290-301Crossref PubMed Scopus (14) Google Scholar). The apparentK m values for NADH (K mNADH), ferricyanide (K mFe), and recombinant Pb5 (K mPb5) and the catalytic constants (k catNADH,k catFe, andk catPb5) were evaluated by direct curve fitting of the data according to the Michaelis-Menten equation. The k catNADH,k catFe, andk catPb5 values were determined from the experiments to evaluate theK mNADH,K mFe, andK mPb5 values, respectively. The kinetic data were measured in 10 mmpotassium phosphate (pH 7.0) at 25 °C. For the measurement of theK mNADH values, 100 μm potassium ferricyanide was used as an electron acceptor. For the measurements of theK mFe andK mPb5 values, 300 μm NADH was used as an electron donor. The reduction rate of ferricyanide was measured at 420 nm using a molar extinction coefficient of 1.02 × 103m−1 cm−1. The reduction rate of recombinant Pb5 was measured at 556 nm using a difference in molar extinction coefficient of natural Pb5 between the oxidized and reduced states, 1.9 × 104m−1cm−1 (43Omura T. Takesue S. J. Biochem. (Tokyo). 1970; 67: 249-257Crossref PubMed Scopus (656) Google Scholar). The concentration of NADH was determined using a molar extinction coefficient, 6.3 × 103m−1 cm−1 at 340 nm. Rapid reaction was analyzed with a Photal RA-401 stopped flow spectrophotometer (Otsuka Electronics) equipped with a Lauda RMS thermostatically regulated circulating water bath in 10 mm potassium phosphate (pH 7.0) at 25 °C. The spectral changes of enzymes during and after the electron transfer from NADH to ferricyanide were analyzed as follows. Equal volumes of the enzyme solution containing potassium ferricyanide and the NADH solution were rapidly mixed, and the rapid scan spectra at 460–800 nm and the time courses of the absorbance changes at 460, 530, and 620 nm were measured. The initial concentrations of NADH, oxidized enzyme, and potassium ferricyanide in the reaction mixtures were 1 mm, 10 μm, and 150 μm, respectively. Under these experimental conditions, a turnover phase and a subsequent reduction phase after the consumption of ferricyanide were observed. The rate constant for the absorbance change after the turnover (k) was determined by single exponential curve fitting of the data as previously described (33Kimura S. Emi Y. Ikushiro S. Iyanagi T. Biochim. Biophys. Acta. 1999; 1430: 290-301Crossref PubMed Scopus (14) Google Scholar). Spectral changes of the enzymes during the reduction with NADH were analyzed with rapid scan spectra and time courses of the absorbance changes at 460 nm in the presence and absence of 1 mmNAD+. The enzyme solution, which contains or does not contain NAD+, was rapidly mixed with the NADH solution. The rapid scan spectra in the region at 420–560 and at 560–730 nm were measured separately and were joined at 560 nm. In the measurement of the rapid scan spectra, the gate time was 4 ms. The rate constants of reduction (k red) in the presence (k red+NAD) and absence of 1 mm NAD+(k red−NAD) were determined by single exponential curve fitting of the data at 460 nm. The flavin cofactor in the enzymes was photoreduced in an anaerobic cuvette containing 40 μmprotein, 1 μm 5-deazariboflavin, 5 mm EDTA, 200 μm NAD+, ∼0.5 μmindigodisulfonate, and 10 mm potassium phosphate (pH 7.0). The solutions were made anaerobic by successive flushing with oxygen-free argon gas with gentle agitation for more than 50 min. The absorption spectra were observed before and after illumination at 25 °C with a 300 W halogen lamp at room temperature. The dissociation constant of the oxidized enzyme for NAD+(K dNAD+) was determined by measuring the perturbation of the flavin spectrum as previously described (28Kimura S. Nishida H. Iyanagi T. J. Biochem. (Tokyo). 2001; 130: 481-490Crossref PubMed Scopus (25) Google Scholar). Approximately 40 μm of oxidized mutant proteins were titrated with NAD+ in 10 mm potassium phosphate (pH 7.0) at 25 °C. After the successive addition of NAD+ into both the sample and reference cells, the absorption spectra were measured. TheK dNAD+ values were determined by direct curve fitting of the difference in the absorbance at the wavelength, where the absorbance change caused by the addition of NAD+ was largest in the 400–550-nm region, with the theoretical equation for a 1:1 binding mechanism taking into account the dilution. All of the purified mutant Pb5Rs showed a single band on an SDS-PAGE gel that was located at the same position as the WT. The yields of the purified T66S, T66A, and T66V mutants from 1 liter of culture fluid were 15.7, 20.3, and 14.5 mg, respectively. In all mutant proteins, the only bound flavin detected on the TLC plate was FAD. The molar extinction coefficients of the T66S, T66A, and T66V mutants at 460 nm were similar to that of the WT and were 1.05 × 104, 1.01 × 104, and 1.06 × 104m−1 cm−1, respectively. The absorption spectrum of the T66S mutant was almost identical to that of the WT (Fig. 2 A, panel a). The absorption spectra of the T66A and T66V mutants were also similar to that of the WT, but slight spectral changes were observed (Fig. 2 A, panels b and c). The absorption spectrum of the T66V mutant was blue-shifted by ∼3 nm in comparison with that of the WT. The intensities of the peaks at 390 nm were slightly decreased in the spectra of the T66A and T66V mutants. The CD spectra of the T66A, T66S, and T66V mutants were also similar to that of the WT (Fig. 2 B). All of the mutant proteins showed a significant decrease in the intensity of the fluorescence emission of FAD. The intensities of the fluorescence emission spectra of free FAD, the WT, and the T66S, T66A, and T66V mutants at 524 nm were 38.9, 1.3, 1.5, 1.4, and 1.3, respectively (spectra are not shown). These data show that the mutations did not change the overall structure of the oxidized form of the enzyme. The apparent steady-state kinetic parameters and the K dNAD+ values are shown in Table I. TheK mNADH values of the T66S, T66A, and T66V mutants were similar to that of the WT, indicating that the mutations in these proteins did not affect the apparent affinity for NADH. Thek catNADH values of the T66S and T66A mutants were almost identical to that of the WT, but that of the T66V mutant was ∼10% of that of the WT. TheK mFe values of the three mutants were similar to that of the WT. The changes in thek catFe values of the three mutants were similar to those in thek catNADH values. Only the mutation of Thr66 to valine significantly affected the electron transfer from NADH to ferricyanide.Table IApparent kinetic parameters evaluated by steady-state analysis and dissociation constants for NAD +EnzymeNADH1-a100 μmferricyanide was used as an electron receptor.Ferricyanide1-b300 μm NADH was used as an electron acceptor.Pb51-b300 μm NADH was used as an electron acceptor.K dNAD+K mNADHk catNADHK mFek catFeK mPb5k catPb5μms −1μms −1μms −1μmWT3.1 ± 0.3" @default.
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• W2145194877 date "2003-02-01" @default.
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• W2145194877 title "Role of Thr66 in Porcine NADH-cytochromeb 5 Reductase in Catalysis and Control of the Rate-limiting Step in Electron Transfer" @default.
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• W2145194877 doi "https://doi.org/10.1074/jbc.m209838200" @default.
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