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• W2066746650 abstract "The molecular masses of the purified, recombinant nucleotide-binding domains (domains I and III) of transhydrogenase from Rhodospirillum rubrum were determined by electrospray mass spectrometry. The values obtained, 40,273 and 21,469 Da, for domains I and III, respectively, are similar to those estimated from the amino acid sequences of the proteins. Evidently, there are no prosthetic groups or metal centers that can serve as reducible intermediates in hydride transfer between nucleotides bound to these proteins. The transient-state kinetics of hydride transfer catalyzed by mixtures of recombinant domains I and III were studied by stopped-flow spectrophotometry. The data indicate that oxidation of NADPH, bound to domain III, and reduction of acetylpyridine adenine dinucleotide (an NAD+ analogue), bound to domain I, are simultaneous and very fast. The transient-state reaction proceeds as a biphasic burst of hydride transfer before establishment of a steady state, which is limited by slow release of NADP+.Hydride transfer between the nucleotides is evidently direct. This conclusion indicates that the nicotinamide rings of the nucleotides are in close apposition during the hydride transfer reaction, and it imposes firm constraints on the mechanism by which transhydrogenation is linked to proton translocation. The molecular masses of the purified, recombinant nucleotide-binding domains (domains I and III) of transhydrogenase from Rhodospirillum rubrum were determined by electrospray mass spectrometry. The values obtained, 40,273 and 21,469 Da, for domains I and III, respectively, are similar to those estimated from the amino acid sequences of the proteins. Evidently, there are no prosthetic groups or metal centers that can serve as reducible intermediates in hydride transfer between nucleotides bound to these proteins. The transient-state kinetics of hydride transfer catalyzed by mixtures of recombinant domains I and III were studied by stopped-flow spectrophotometry. The data indicate that oxidation of NADPH, bound to domain III, and reduction of acetylpyridine adenine dinucleotide (an NAD+ analogue), bound to domain I, are simultaneous and very fast. The transient-state reaction proceeds as a biphasic burst of hydride transfer before establishment of a steady state, which is limited by slow release of NADP+. Hydride transfer between the nucleotides is evidently direct. This conclusion indicates that the nicotinamide rings of the nucleotides are in close apposition during the hydride transfer reaction, and it imposes firm constraints on the mechanism by which transhydrogenation is linked to proton translocation. Transhydrogenase is found in the inner membranes of animal mitochondria and the cytoplasmic membranes of some bacteria. It couples the transfer of hydride ion equivalents between NAD(H) and NADP(H) to the translocation of protons across the membrane. The net reaction is as follows. NADH+NADP++Hout+⇔NAD++NADPH+Hin+Equation 1 For many years, the question as to whether hydride transfer between the nucleotides is direct or indirect has been a matter of controversy. It is central to our understanding of the energy-coupling reactions.Transhydrogenase comprises three domains. Domains I and III protrude from the membrane (on the matrix side in mitochondria and on the cytoplasmic side in bacteria). Domain II spans the membrane. There are separate sites on the enzyme for NAD(H) and for NADP(H); the former is located on domain I, and the latter on domain III (for reviews, see Refs. 1Jackson J.B. J. Bioenerg. Biomembr. 1991; 23: 715-741Crossref PubMed Scopus (89) Google Scholar, 2Olausson T. Fjellstrom O. Meuller J. Rydstrom J. Biochim. Biophys. Acta. 1995; 1231: 1-19Crossref PubMed Scopus (74) Google Scholar, 3Hatefi Y. Yamaguchi M. FASEB J. 1996; 10: 444-452Crossref PubMed Scopus (81) Google Scholar).The results of some early experiments on transhydrogenases from mitochondria and from Rhodospirillum rubrum were interpreted as evidence for the existence of a stable, reduced-enzyme intermediate (4Fisher R.R. Guillory R.J. J. Biol. Chem. 1971; 246: 4687-4693Abstract Full Text PDF PubMed Google Scholar, 5Jacobs E. Fisher R.R. Biochemistry. 1979; 18: 4315-4322Crossref PubMed Scopus (9) Google Scholar, 6Fisher R.R. Earle S.R. Everse J. Anderson B.M. You K.S. The Pyridine Nucleotide Coenzymes. Academic Press, New York1982: 279-324Crossref Google Scholar). It was implied that a functional group on the enzyme, presumably either an amino acid residue or an unidentified prosthetic group, can serve alternately as a hydride acceptor and hydride donor. For example, E+NADH⇔E(H)+NAD+Equation 2 E(H)+NADP+⇔E+NADPHEquation 3 where E(H) represents the reduced-enzyme intermediate. However, in subsequent work other plausible explanations were found for the earlier data (7Lever T.M. Palmer T. Cunningham I.J. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1991; 197: 247-255Crossref PubMed Scopus (40) Google Scholar, 8Stilwell S.N. Bizouarn T. Jackson J.B. Biochim. Biophys. Acta. 1997; 1320: 83-94Crossref PubMed Scopus (18) Google Scholar). Moreover, the conclusions from steady-state kinetic analysis of transhydrogenase from various sources (7Lever T.M. Palmer T. Cunningham I.J. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1991; 197: 247-255Crossref PubMed Scopus (40) Google Scholar, 9Hanson R.L. J. Biol. Chem. 1979; 254: 888-893Abstract Full Text PDF PubMed Google Scholar, 10Homyk M. Bragg P.D. Biochim. Biophys. Acta. 1979; 571: 201-217Crossref PubMed Scopus (28) Google Scholar, 11Enander K. Rydstrom J. J. Biol. Chem. 1982; 257: 14760-14766Abstract Full Text PDF PubMed Google Scholar) have been interpreted as evidence that the reaction proceeds through the formation of a ternary complex of enzyme and nucleotide substrates. The addition of nucleotides is random, and fast, relative to the rate of a subsequent step in turnover. The reaction does not appear to take place via a substituted enzyme mechanism, and therefore the existence of a reduced enzyme intermediate, which is stable in the absence of nucleotide, is unlikely (viz. reactions exemplified by Equations 2 and 3). However, the steady-state data do not rule out the possible existence of a reduced enzyme intermediatewithin the ternary complex, that is a reaction of the following type. E+NADH+NADP+⇔NADH·E·NADP+⇔NAD+·E(H)·NADP+⇔Equation 4 NAD+·E·NADPH⇔E+NAD++NADPHThe possibility that Cys residues in the polypeptide chain might serve as reducible intermediates in hydride transfer (see, for example, Refs. 1Jackson J.B. J. Bioenerg. Biomembr. 1991; 23: 715-741Crossref PubMed Scopus (89) Google Scholar and 12Persson B. Rydstrom J. Biochem. Biophys. Res. Commun. 1987; 142: 573-578Crossref PubMed Scopus (24) Google Scholar) has been eliminated by amino acid sequence comparisons, there are no conserved Cys residues in transhydrogenases from different species, and by the fact that complete Cys replacement has only a minimal effect on transhydrogenation activity (13Meuller J. Hou C. Bragg P.D. Rydstrom J. Biochem. J. 1997; 234: 681-687Crossref Scopus (33) Google Scholar). It is unlikely that other amino acid residues have redox potentials in the appropriate range to serve as intermediates in transhydrogenation between NAD(H) and NADP(H). It has sometimes been stated in the literature that transhydrogenase is devoid of prosthetic groups that might be involved in the hydride transfer pathway, although our survey indicates that studies are incomplete. 1) It was established many years ago that there is no detectable flavin fluorescence from purified preparations of the mitochondrial enzyme (14Hojeberg B. Rydstrom J. Methods Enzymol. 1979; 55: 275-283Crossref PubMed Scopus (2) Google Scholar). 2) Analyses of amino acid sequences of transhydrogenases do not reveal the existence of metal-binding motifs. 3) It has been shown that concentrated solutions of the highly purified recombinant domains I and III (which together are catalytically active, see below) do not have any absorbance that might be attributable to chromophoric groups in the proteins (15Diggle C. Hutton M. Jones G.R. Thomas C.M. Jackson J.B. Eur. J. Biochem. 1995; 228: 719-726Crossref PubMed Scopus (59) Google Scholar, 16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar). However, we are unaware of studies on transhydrogenase which rule out the presence of weakly, or nonabsorbing, prosthetic groups. By analogy with dehydrogenases, a number of possible prosthetic groups or metal ions might be envisaged to have a role as an intermediate in hydride transfer. For example, in view of the strong sequence similarity between transhydrogenase and the soluble enzyme alanine dehydrogenase (17Cunningham I.J. Williams R. Palmer T. Thomas C.M. Jackson J.B. Biochim. Biophys. Acta. 1992; 1100: 332-338Crossref PubMed Scopus (37) Google Scholar), covalently bound pyruvate might be considered as a potential hydride acceptor.A mixture of the isolated recombinant forms of domains I and III ofR. rubrum transhydrogenase catalyzes the so-called “cyclic reaction” at a rate approaching that observed with the complete, membrane-located enzyme (16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar, 18Yamaguchi M. Hatefi Y. Biochim. Biophys. Acta. 1997; 1318: 225-234Crossref PubMed Scopus (36) Google Scholar). Evidently the complex of domains I and III is capable of rapid rates of hydride transfer, even in the absence of membrane-spanning domain II, and thus the apparatus for hydride transfer is located entirely within the two peripheral domains. We report below on the use of electrospray mass spectrometry to determine accurately the molecular masses of these peripheral domains with a view to establishing whether or not they possess covalently bound groups that might participate in the hydride transfer reaction. We also describe an experiment in which we examine the pre-steady-state kinetics of transhydrogenation catalyzed by a mixture of recombinant domains I and III using stopped-flow spectroscopy. In contrast to steady-state kinetic analysis, this procedure can reveal the presence of reaction intermediates. There are no other published descriptions of the pre-steady-state kinetics of reactions catalyzed by transhydrogenase. As hydride donor (binding to domain III), we use NADPH, and as hydride acceptor (binding to domain I), we use the NAD+ analogue, AcPdAD+. 1The abbreviations used are: AcPdAD+, acetylpyridine adenine dinucleotide. 1The abbreviations used are: AcPdAD+, acetylpyridine adenine dinucleotide. The difference in the absorbance spectra between the reduced forms of the two nucleotides enables us to measure the rate of oxidation of NADPH, and the rate of reduction of AcPdAD+, in real time.MATERIALS AND METHODSRecombinant forms of domain I and domain III of R. rubrum transhydrogenase were expressed in E. coli C600 from plasmids pCD1 (15Diggle C. Hutton M. Jones G.R. Thomas C.M. Jackson J.B. Eur. J. Biochem. 1995; 228: 719-726Crossref PubMed Scopus (59) Google Scholar) and pCD2 (16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar), respectively, and purified by column chromatography (15Diggle C. Hutton M. Jones G.R. Thomas C.M. Jackson J.B. Eur. J. Biochem. 1995; 228: 719-726Crossref PubMed Scopus (59) Google Scholar, 16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar, 17Cunningham I.J. Williams R. Palmer T. Thomas C.M. Jackson J.B. Biochim. Biophys. Acta. 1992; 1100: 332-338Crossref PubMed Scopus (37) Google Scholar). The purity was confirmed by SDS-polyacrylamide gel electrophoresis (7Lever T.M. Palmer T. Cunningham I.J. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1991; 197: 247-255Crossref PubMed Scopus (40) Google Scholar), and the protein concentrations were determined using the microtannin assay (19Mejbaum-Katzenellenbogen S. Drobryszycka W.J. Clin. Chem. Acta. 1959; 4: 515-522Crossref Scopus (156) Google Scholar). As normally prepared, the domain III protein is associated with tightly bound NADP+ (typically 0.1–0.4 mol·mol−1) and NADPH (about 0.5 mol·mol−1) (16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar). For stopped flow experiments the bound NADP+ was replaced by incubating domain III protein (75 μm) in 10 mm(NH4)2SO4, 20 mm Hepes buffer, pH 8.0, with 105 μm NADPH at 4 °C for 2 h (16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar).Stopped-flow experiments were performed with an Applied Photophysics DX-17MV in its absorbance mode (2-mm optical path length). The instrument dead time, determined from the reaction ofl-ascorbic acid with 2,6-dichlorophenolindophenol, as described (20Tonomura B. Nakatani H. Ohnishi M. Yamaguchi-Ito J. Hiromi K. Anal. Biochem. 1978; 84: 370-383Crossref PubMed Scopus (285) Google Scholar), was 1.31 ms. The monochromator slit widths were set to 5 nm. The absorbance coefficient at 375 nm for AcPdAD+reduction (corrected for the contribution from accompanying NADPH oxidation) was 6.1 mm−1·cm−1(21Palmer T. Jackson J.B. Biochim. Biophys. Acta. 1992; 1099: 157-162Crossref PubMed Scopus (31) Google Scholar) and that at 320 nm for NADPH oxidation (corrected for the contribution from accompanying AcPdAD+ reduction) was 3.01 mm−1·cm−1 (calculated from data given (22Siegel J.M. Montgomery G.A. Bock R.M. Arch. Biochem. Biophys. 1959; 82: 288-299Crossref PubMed Scopus (98) Google Scholar)). Rate constants and amplitudes were calculated from the raw data using the instrument software, making due correction for the apparatus dead time.Samples of domains I and III were prepared for analysis by electrospray mass spectrometry by dialyzing samples (25 μm) against water. The examination of domain I was performed in collaboration with Dr. C. Robinson, University of Oxford, using a Micromass Platform II and Nanoflow electrospray ionization. Data were acquired using MassLynx software. The domain III analysis was carried out in collaboration with Dr. P. R. Ashton, University of Birmingham, using a VG-Prospec in electrospray ionization mode. In both cases the carrier matrix was 20% methanol. The precision of both instruments is better than ±0.01%. Molecular masses were estimated from the amino acid sequences of domains I and III using the Genetics Computer Group program (University of Wisconsin). Transhydrogenase is found in the inner membranes of animal mitochondria and the cytoplasmic membranes of some bacteria. It couples the transfer of hydride ion equivalents between NAD(H) and NADP(H) to the translocation of protons across the membrane. The net reaction is as follows. NADH+NADP++Hout+⇔NAD++NADPH+Hin+Equation 1 For many years, the question as to whether hydride transfer between the nucleotides is direct or indirect has been a matter of controversy. It is central to our understanding of the energy-coupling reactions. Transhydrogenase comprises three domains. Domains I and III protrude from the membrane (on the matrix side in mitochondria and on the cytoplasmic side in bacteria). Domain II spans the membrane. There are separate sites on the enzyme for NAD(H) and for NADP(H); the former is located on domain I, and the latter on domain III (for reviews, see Refs. 1Jackson J.B. J. Bioenerg. Biomembr. 1991; 23: 715-741Crossref PubMed Scopus (89) Google Scholar, 2Olausson T. Fjellstrom O. Meuller J. Rydstrom J. Biochim. Biophys. Acta. 1995; 1231: 1-19Crossref PubMed Scopus (74) Google Scholar, 3Hatefi Y. Yamaguchi M. FASEB J. 1996; 10: 444-452Crossref PubMed Scopus (81) Google Scholar). The results of some early experiments on transhydrogenases from mitochondria and from Rhodospirillum rubrum were interpreted as evidence for the existence of a stable, reduced-enzyme intermediate (4Fisher R.R. Guillory R.J. J. Biol. Chem. 1971; 246: 4687-4693Abstract Full Text PDF PubMed Google Scholar, 5Jacobs E. Fisher R.R. Biochemistry. 1979; 18: 4315-4322Crossref PubMed Scopus (9) Google Scholar, 6Fisher R.R. Earle S.R. Everse J. Anderson B.M. You K.S. The Pyridine Nucleotide Coenzymes. Academic Press, New York1982: 279-324Crossref Google Scholar). It was implied that a functional group on the enzyme, presumably either an amino acid residue or an unidentified prosthetic group, can serve alternately as a hydride acceptor and hydride donor. For example, E+NADH⇔E(H)+NAD+Equation 2 E(H)+NADP+⇔E+NADPHEquation 3 where E(H) represents the reduced-enzyme intermediate. However, in subsequent work other plausible explanations were found for the earlier data (7Lever T.M. Palmer T. Cunningham I.J. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1991; 197: 247-255Crossref PubMed Scopus (40) Google Scholar, 8Stilwell S.N. Bizouarn T. Jackson J.B. Biochim. Biophys. Acta. 1997; 1320: 83-94Crossref PubMed Scopus (18) Google Scholar). Moreover, the conclusions from steady-state kinetic analysis of transhydrogenase from various sources (7Lever T.M. Palmer T. Cunningham I.J. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1991; 197: 247-255Crossref PubMed Scopus (40) Google Scholar, 9Hanson R.L. J. Biol. Chem. 1979; 254: 888-893Abstract Full Text PDF PubMed Google Scholar, 10Homyk M. Bragg P.D. Biochim. Biophys. Acta. 1979; 571: 201-217Crossref PubMed Scopus (28) Google Scholar, 11Enander K. Rydstrom J. J. Biol. Chem. 1982; 257: 14760-14766Abstract Full Text PDF PubMed Google Scholar) have been interpreted as evidence that the reaction proceeds through the formation of a ternary complex of enzyme and nucleotide substrates. The addition of nucleotides is random, and fast, relative to the rate of a subsequent step in turnover. The reaction does not appear to take place via a substituted enzyme mechanism, and therefore the existence of a reduced enzyme intermediate, which is stable in the absence of nucleotide, is unlikely (viz. reactions exemplified by Equations 2 and 3). However, the steady-state data do not rule out the possible existence of a reduced enzyme intermediatewithin the ternary complex, that is a reaction of the following type. E+NADH+NADP+⇔NADH·E·NADP+⇔NAD+·E(H)·NADP+⇔Equation 4 NAD+·E·NADPH⇔E+NAD++NADPHThe possibility that Cys residues in the polypeptide chain might serve as reducible intermediates in hydride transfer (see, for example, Refs. 1Jackson J.B. J. Bioenerg. Biomembr. 1991; 23: 715-741Crossref PubMed Scopus (89) Google Scholar and 12Persson B. Rydstrom J. Biochem. Biophys. Res. Commun. 1987; 142: 573-578Crossref PubMed Scopus (24) Google Scholar) has been eliminated by amino acid sequence comparisons, there are no conserved Cys residues in transhydrogenases from different species, and by the fact that complete Cys replacement has only a minimal effect on transhydrogenation activity (13Meuller J. Hou C. Bragg P.D. Rydstrom J. Biochem. J. 1997; 234: 681-687Crossref Scopus (33) Google Scholar). It is unlikely that other amino acid residues have redox potentials in the appropriate range to serve as intermediates in transhydrogenation between NAD(H) and NADP(H). It has sometimes been stated in the literature that transhydrogenase is devoid of prosthetic groups that might be involved in the hydride transfer pathway, although our survey indicates that studies are incomplete. 1) It was established many years ago that there is no detectable flavin fluorescence from purified preparations of the mitochondrial enzyme (14Hojeberg B. Rydstrom J. Methods Enzymol. 1979; 55: 275-283Crossref PubMed Scopus (2) Google Scholar). 2) Analyses of amino acid sequences of transhydrogenases do not reveal the existence of metal-binding motifs. 3) It has been shown that concentrated solutions of the highly purified recombinant domains I and III (which together are catalytically active, see below) do not have any absorbance that might be attributable to chromophoric groups in the proteins (15Diggle C. Hutton M. Jones G.R. Thomas C.M. Jackson J.B. Eur. J. Biochem. 1995; 228: 719-726Crossref PubMed Scopus (59) Google Scholar, 16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar). However, we are unaware of studies on transhydrogenase which rule out the presence of weakly, or nonabsorbing, prosthetic groups. By analogy with dehydrogenases, a number of possible prosthetic groups or metal ions might be envisaged to have a role as an intermediate in hydride transfer. For example, in view of the strong sequence similarity between transhydrogenase and the soluble enzyme alanine dehydrogenase (17Cunningham I.J. Williams R. Palmer T. Thomas C.M. Jackson J.B. Biochim. Biophys. Acta. 1992; 1100: 332-338Crossref PubMed Scopus (37) Google Scholar), covalently bound pyruvate might be considered as a potential hydride acceptor. A mixture of the isolated recombinant forms of domains I and III ofR. rubrum transhydrogenase catalyzes the so-called “cyclic reaction” at a rate approaching that observed with the complete, membrane-located enzyme (16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar, 18Yamaguchi M. Hatefi Y. Biochim. Biophys. Acta. 1997; 1318: 225-234Crossref PubMed Scopus (36) Google Scholar). Evidently the complex of domains I and III is capable of rapid rates of hydride transfer, even in the absence of membrane-spanning domain II, and thus the apparatus for hydride transfer is located entirely within the two peripheral domains. We report below on the use of electrospray mass spectrometry to determine accurately the molecular masses of these peripheral domains with a view to establishing whether or not they possess covalently bound groups that might participate in the hydride transfer reaction. We also describe an experiment in which we examine the pre-steady-state kinetics of transhydrogenation catalyzed by a mixture of recombinant domains I and III using stopped-flow spectroscopy. In contrast to steady-state kinetic analysis, this procedure can reveal the presence of reaction intermediates. There are no other published descriptions of the pre-steady-state kinetics of reactions catalyzed by transhydrogenase. As hydride donor (binding to domain III), we use NADPH, and as hydride acceptor (binding to domain I), we use the NAD+ analogue, AcPdAD+. 1The abbreviations used are: AcPdAD+, acetylpyridine adenine dinucleotide. 1The abbreviations used are: AcPdAD+, acetylpyridine adenine dinucleotide. The difference in the absorbance spectra between the reduced forms of the two nucleotides enables us to measure the rate of oxidation of NADPH, and the rate of reduction of AcPdAD+, in real time. MATERIALS AND METHODSRecombinant forms of domain I and domain III of R. rubrum transhydrogenase were expressed in E. coli C600 from plasmids pCD1 (15Diggle C. Hutton M. Jones G.R. Thomas C.M. Jackson J.B. Eur. J. Biochem. 1995; 228: 719-726Crossref PubMed Scopus (59) Google Scholar) and pCD2 (16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar), respectively, and purified by column chromatography (15Diggle C. Hutton M. Jones G.R. Thomas C.M. Jackson J.B. Eur. J. Biochem. 1995; 228: 719-726Crossref PubMed Scopus (59) Google Scholar, 16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar, 17Cunningham I.J. Williams R. Palmer T. Thomas C.M. Jackson J.B. Biochim. Biophys. Acta. 1992; 1100: 332-338Crossref PubMed Scopus (37) Google Scholar). The purity was confirmed by SDS-polyacrylamide gel electrophoresis (7Lever T.M. Palmer T. Cunningham I.J. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1991; 197: 247-255Crossref PubMed Scopus (40) Google Scholar), and the protein concentrations were determined using the microtannin assay (19Mejbaum-Katzenellenbogen S. Drobryszycka W.J. Clin. Chem. Acta. 1959; 4: 515-522Crossref Scopus (156) Google Scholar). As normally prepared, the domain III protein is associated with tightly bound NADP+ (typically 0.1–0.4 mol·mol−1) and NADPH (about 0.5 mol·mol−1) (16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar). For stopped flow experiments the bound NADP+ was replaced by incubating domain III protein (75 μm) in 10 mm(NH4)2SO4, 20 mm Hepes buffer, pH 8.0, with 105 μm NADPH at 4 °C for 2 h (16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar).Stopped-flow experiments were performed with an Applied Photophysics DX-17MV in its absorbance mode (2-mm optical path length). The instrument dead time, determined from the reaction ofl-ascorbic acid with 2,6-dichlorophenolindophenol, as described (20Tonomura B. Nakatani H. Ohnishi M. Yamaguchi-Ito J. Hiromi K. Anal. Biochem. 1978; 84: 370-383Crossref PubMed Scopus (285) Google Scholar), was 1.31 ms. The monochromator slit widths were set to 5 nm. The absorbance coefficient at 375 nm for AcPdAD+reduction (corrected for the contribution from accompanying NADPH oxidation) was 6.1 mm−1·cm−1(21Palmer T. Jackson J.B. Biochim. Biophys. Acta. 1992; 1099: 157-162Crossref PubMed Scopus (31) Google Scholar) and that at 320 nm for NADPH oxidation (corrected for the contribution from accompanying AcPdAD+ reduction) was 3.01 mm−1·cm−1 (calculated from data given (22Siegel J.M. Montgomery G.A. Bock R.M. Arch. Biochem. Biophys. 1959; 82: 288-299Crossref PubMed Scopus (98) Google Scholar)). Rate constants and amplitudes were calculated from the raw data using the instrument software, making due correction for the apparatus dead time.Samples of domains I and III were prepared for analysis by electrospray mass spectrometry by dialyzing samples (25 μm) against water. The examination of domain I was performed in collaboration with Dr. C. Robinson, University of Oxford, using a Micromass Platform II and Nanoflow electrospray ionization. Data were acquired using MassLynx software. The domain III analysis was carried out in collaboration with Dr. P. R. Ashton, University of Birmingham, using a VG-Prospec in electrospray ionization mode. In both cases the carrier matrix was 20% methanol. The precision of both instruments is better than ±0.01%. Molecular masses were estimated from the amino acid sequences of domains I and III using the Genetics Computer Group program (University of Wisconsin). Recombinant forms of domain I and domain III of R. rubrum transhydrogenase were expressed in E. coli C600 from plasmids pCD1 (15Diggle C. Hutton M. Jones G.R. Thomas C.M. Jackson J.B. Eur. J. Biochem. 1995; 228: 719-726Crossref PubMed Scopus (59) Google Scholar) and pCD2 (16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar), respectively, and purified by column chromatography (15Diggle C. Hutton M. Jones G.R. Thomas C.M. Jackson J.B. Eur. J. Biochem. 1995; 228: 719-726Crossref PubMed Scopus (59) Google Scholar, 16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar, 17Cunningham I.J. Williams R. Palmer T. Thomas C.M. Jackson J.B. Biochim. Biophys. Acta. 1992; 1100: 332-338Crossref PubMed Scopus (37) Google Scholar). The purity was confirmed by SDS-polyacrylamide gel electrophoresis (7Lever T.M. Palmer T. Cunningham I.J. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1991; 197: 247-255Crossref PubMed Scopus (40) Google Scholar), and the protein concentrations were determined using the microtannin assay (19Mejbaum-Katzenellenbogen S. Drobryszycka W.J. Clin. Chem. Acta. 1959; 4: 515-522Crossref Scopus (156) Google Scholar). As normally prepared, the domain III protein is associated with tightly bound NADP+ (typically 0.1–0.4 mol·mol−1) and NADPH (about 0.5 mol·mol−1) (16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar). For stopped flow experiments the bound NADP+ was replaced by incubating domain III protein (75 μm) in 10 mm(NH4)2SO4, 20 mm Hepes buffer, pH 8.0, with 105 μm NADPH at 4 °C for 2 h (16Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar). Stopped-flow experiments were performed with an Applied Photophysics DX-17MV in its absorbance mode (2-mm optical path length). The instrument dead time, determined from the reaction ofl-ascorbic acid with 2,6-dichlorophenolindophenol, as described (20Tonomura B. Nakatani H. Ohnishi M. Yamaguchi-Ito J. Hiromi K. Anal. Biochem. 1978; 84: 370-383Crossref PubMed Scopus (285) Google Scholar), was 1.31 ms. The monochromator slit widths were set to 5 nm. The absorbance coefficient at 375 nm for AcPdAD+reduction (corrected for the contribution from accompanying NADPH oxidation) was 6.1 mm−1·cm−1(21Palmer T. Jackson J.B. Biochim. Biophys. Acta. 1992; 1099: 157-162Crossref PubMed Scopus (31) Google Scholar) and that at 320 nm for NADPH oxidation (corrected for the contribution from accompanying AcPdAD+ reduction) was 3.01 mm−1·cm−1 (calculated from data given (22Siegel J.M. Montgomery G.A. Bock R.M. Arch. Biochem. Biophys. 1959; 82: 288-299Crossref PubMed Scopus (98) Google Scholar)). Rate constants and amplitudes were calculated from the raw data using the instrument software, making due correction for the apparatus dead time. Samples of domains I and III were prepared for analysis by electrospray mass spectrometry by dialyzing samples (25 μm) against water. The examination of domain I was performed in collaboration with Dr. C. Robinson, University of Oxford, using a Micromass Platform II and Nanoflow electrospray ionization. Data were acquired using MassLynx software. The domain III analysis was carried out in collaboration with Dr. P. R. Ashton, University of Birmingham, using a VG-Prospec in electrospray ionization mode. In both cases the carrier matrix was 20% methanol. The precision of both instruments is better than ±0.01%. Molecular masses were estimated from the amino acid sequences of domains I and III using the Genetics Computer Group program (University of Wisconsin). We are very grateful to Carol Robinson and Peter Ashton for their help with the mass spectrometry analysis." @default.
• W2066746650 created "2016-06-24" @default.
• W2066746650 creator A5033840732 @default.
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• W2066746650 title "Evidence That the Transfer of Hydride Ion Equivalents between Nucleotides by Proton-translocating Transhydrogenase Is Direct" @default.
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