Interactions between Transhydrogenase and Thio-nicotinamide Analogues of NAD(H) and NADP(H) Underline the Importance of Nucleotide Conformational Changes in Coupling to Proton Translocation
2003; Elsevier BV; Volume: 278; Issue: 35 Linguagem: Inglês
10.1074/jbc.m303061200
ISSN1083-351X
AutoresAvtar Singh, Jamie D. Venning, Philip G. Quirk, Gijs I. van Boxel, Daniel J. Rodrigues, Scott A. White, J. Baz Jackson,
Tópico(s)ATP Synthase and ATPases Research
ResumoTranshydrogenase couples the reduction of NADP+ by NADH to inward proton translocation across mitochondrial and bacterial membranes. The coupling reactions occur within the protein by long distance conformational changes. In intact transhydrogenase and in complexes formed from the isolated, nucleotide-binding components, thio-NADP(H) is a good analogue for NADP(H), but thio-NAD(H) is a poor analogue for NAD(H). Crystal structures of the nucleotide-binding components show that the twists of the 3-carbothiamide groups of thio-NADP+ and of thio-NAD+ (relative to the planes of the pyridine rings), which are defined by the dihedral, X am, are altered relative to the twists of the 3-carboxamide groups of the physiological nucleotides. The finding that thio-NADP+ is a good substrate despite an increased X am value shows that approach of the NADH prior to hydride transfer is not obstructed by the S atom in the analogue. That thio-NAD(H) is a poor substrate appears to be the result of failure in the conformational change that establishes the ground state for hydride transfer. This might be a consequence of restricted rotation of the 3-carbothiamide group during the conformational change. Transhydrogenase couples the reduction of NADP+ by NADH to inward proton translocation across mitochondrial and bacterial membranes. The coupling reactions occur within the protein by long distance conformational changes. In intact transhydrogenase and in complexes formed from the isolated, nucleotide-binding components, thio-NADP(H) is a good analogue for NADP(H), but thio-NAD(H) is a poor analogue for NAD(H). Crystal structures of the nucleotide-binding components show that the twists of the 3-carbothiamide groups of thio-NADP+ and of thio-NAD+ (relative to the planes of the pyridine rings), which are defined by the dihedral, X am, are altered relative to the twists of the 3-carboxamide groups of the physiological nucleotides. The finding that thio-NADP+ is a good substrate despite an increased X am value shows that approach of the NADH prior to hydride transfer is not obstructed by the S atom in the analogue. That thio-NAD(H) is a poor substrate appears to be the result of failure in the conformational change that establishes the ground state for hydride transfer. This might be a consequence of restricted rotation of the 3-carbothiamide group during the conformational change. Transhydrogenase is found in the inner membrane of animal mitochondria and in the cytoplasmic membrane of bacteria. The enzyme provides NADPH for biosynthesis and for reduction of glutathione, and in some mammalian tissues, it probably participates in the regulation of flux through the tricarboxylic acid cycle (1Rydstrom J. Hoek J.B. Biochem. J. 1988; 254: 1-10Crossref PubMed Scopus (290) Google Scholar, 2Sazanov L.A. Jackson J.B. FEBS Lett. 1994; 344: 109-116Crossref PubMed Scopus (174) Google Scholar). Under most physiological conditions transhydrogenase is driven in the “forward” direction by the proton electrochemical gradient (Δp) generated by respiratory (or photosynthetic) electron transport. NADH+NADP++Hout↔NAD++NADPH+Hin+(Eq. 1) There is general agreement that coupling between the redox reaction and proton translocation is mediated by changes in protein conformation, although the character of these conformational changes is not known (reviewed in Refs. 3Jackson J.B. White S.A. Quirk P.G. Venning J.D. Biochemistry. 2002; 41: 4173-4185Crossref PubMed Scopus (46) Google Scholar, 4Bizouarn T. Fjellstrom O. Meuller J. Axelsson M. Bergkvist A. Johansson C. Karlsson G. Rydstrom J. Biochim. Biophys. Acta. 2000; 1457: 211-218Crossref PubMed Scopus (56) Google Scholar, 5Hatefi Y. Yamaguchi M. FASEB J. 1996; 10: 444-452Crossref PubMed Scopus (81) Google Scholar). Coupling mechanisms that involve large conformational changes operating over considerable distances are emerging as a common feature in proteins that translocate solutes/ions across membranes, and the amenable properties of transhydrogenase make it an attractive model in the search for fundamental principles. The enzyme has three components. The dI component, which binds NAD+ and NADH, and the dIII component, which binds NADP+ and NADPH, are extrinsic proteins protruding from the membrane (on the matrix side in mitochondria and on the cytoplasmic side in bacteria), and dII spans the membrane. The enzyme is essentially a “dimer” of two dI-dII-dIII “monomers,” although the polypeptide composition is variable among species. Crystal structures of Rhodospirillum rubrum dI (6Buckley P.A. Jackson J.B. Schneider T. White S.A. Rice D.W. Baker P.J. Structure. 2000; 8: 809-815Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 7Prasad G.S. Wahlberg M. Sridhar V. Sundaresan V. Yamaguchi M. Hatefi Y. Stout C.D. Biochemistry. 2002; 41: 12745-12754Crossref PubMed Scopus (35) Google Scholar), bovine dIII (8Prasad G.S. Sridhar V. Yamaguchi M. Hatefi Y. Stout C.D. Nat. Struct. Biol. 1999; 6: 1126-1131Crossref PubMed Scopus (67) Google Scholar), human dIII (9Jackson J.B. Peake S.J. White S.A. FEBS Lett. 1999; 464: 1-8Crossref PubMed Scopus (47) Google Scholar, 10White S.A. Peake S.J. McSweeney S. Leonard G. Cotton N.N.J. Jackson J.B. Structure. 2000; 8: 1-12Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar), and R. rubrum dI2dIII1 complex (11Cotton N.P.J. White S.A. Peake S.J. McSweeney S. Jackson J.B. Structure. 2001; 9: 165-176Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 12van Boxel G.I. Quirk P. Cotton N.J.P. White S.A. Jackson J.B. Biochemistry. 2003; 42: 1217-1226Crossref PubMed Scopus (12) Google Scholar), and an NMR structure of R. rubrum dIII (13Jeeves M. Smith K.J. Quirk P.G. Cotton N.P.J. Jackson J.B. Biochim. Biophys. Acta. 2000; 1459: 248-257Crossref PubMed Scopus (33) Google Scholar) have recently been published. Studies on the transient state kinetics of transhydrogenation reveal that the redox reaction between the two nucleotides is direct (14Venning J.D. Grimley R.L. Bizouarn T. Cotton N.P.J. Jackson J.B. J. Biol. Chem. 1997; 272: 27535-27538Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar, 15Venning J.D. Bizouarn T. Cotton N.P.J. Quirk P.G. Jackson J.B. Eur. J. Biochem. 1998; 257: 202-209Crossref PubMed Scopus (33) Google Scholar). Thus, the nicotinamide and dihydronicotinamide groups are brought into apposition to allow transfer of a hydride ion equivalent between the C-4 positions of the rings. The reaction is stereo-specific for the pro-R (A-side) of NAD(H) and the pro-S (B-side) of NADP(H) (16Lee C.P. Simard-Duquesne N. Ernster L. Hoberman H.D. Biochim. Biophys. Acta. 1965; 105: 397-409Crossref PubMed Google Scholar, 17Fisher R.R. Guillory R.J. J. Biol. Chem. 1971; 246: 4687-4693Abstract Full Text PDF PubMed Google Scholar). Recent interpretations of kinetic and structural work on transhydrogenase have focused on the importance of conformational changes in the nucleotides as well as in the protein (3Jackson J.B. White S.A. Quirk P.G. Venning J.D. Biochemistry. 2002; 41: 4173-4185Crossref PubMed Scopus (46) Google Scholar). It may be possible to test these interpretations through experiments using nucleotide analogues. Thio-NAD(H) and thio-NADP(H), which have a 3-carbothiamide substituent in place of the 3-carboxamide of the pyridine/dihydropyridine ring, have been used extensively in the study of soluble “dehydrogenases” (18Woenchhaus C. Jeck R. Dolphin D. Poulson R. Avramovic O. in Pyridine Nucleotide Coenzymes. John Wiley & Sons, Inc., New York1987: 449-568Google Scholar). The binding properties of the analogues can be different relative to those of the physiological nucleotides and the catalytic rate can be affected; both increases and decreases have been observed in different enzymes (19Cook P.F. Bertagnolli B.L. Dolphin D. Poulson R. Avramovic O. Pyridine Nucleotide Coenzymes. John Wiley & Sons, New York1987: 405-448Google Scholar). An x-ray structure of dihydrofolate reductase (DHFR) 1The abbreviations used are: DHFR, dihydrofolate reductase; thio-NADP+, 3-carbothiamide derivative of NADP+; AcPdAD+, 3-acetylpyridine adenine dinucleotide; Mops, 4-morpholinepropanesulfonic acid.1The abbreviations used are: DHFR, dihydrofolate reductase; thio-NADP+, 3-carbothiamide derivative of NADP+; AcPdAD+, 3-acetylpyridine adenine dinucleotide; Mops, 4-morpholinepropanesulfonic acid. with bound thio-NADP+ showed how small distortions of the nucleotide conformation can lead to pronounced effects on catalysis (20McTigue M.A. Davies J.F. Kaufman B.T. Kraut J. Biochemistry. 1993; 32: 6855-6862Crossref PubMed Scopus (26) Google Scholar). Because the absorbance band of its reduced form is redshifted relative to that of the physiological substrate (and therefore has minimal spectral overlap with NADH), thio-NADP+ has often been used to monitor the activity of proton-translocating transhydrogenase (21Rydstrom J. Methods Enzymol. 1979; 55: 261-275Crossref PubMed Scopus (37) Google Scholar), but few experiments have been carried out using thio-nicotinamide analogues with a view to elucidating mechanistic details of the enzyme. In this report we compare hydride transfer rates to thio-NAD+ and to thio-NADP+ with those to NAD+ and NADP+, respectively, in both the intact R. rubrum transhydrogenase and in dI2dIII1 complexes. It emerges that thio-NAD+ is a poor substrate in the dI site, but thio-NADP+ is a good substrate in the dIII site. To attempt to explain these differences, we have solved the crystal structures of human dIII in its thio-NADP+ form (for comparison with dIII.NADP+; Protein Data Base 1DJL) and of the R. rubrum dI2dIII1 complex loaded with thio-NAD+ and NADP+ (for comparison with complex loaded with NAD+ and NADP+; Protein Data Base 1HZZ). The increased van der Waals' radius of the S atom in the carbothiamide group (1.9 Å compared with 1.4 Å of the O atom) and the increased length of the C = S bond (1.65 Å compared with 1.25 for C = O) have only quite subtle effects on the conformation of the bound nucleotides and the arrangement of the side chains of invariant amino acids at the binding site. We explain the results in terms of the structural changes at the catalytic center that are required to bring together the nicotinamide and dihydronicotinamide rings during hydride transfer, and we discuss the conclusions in the context of the suggestion that these structural changes are associated with proton translocation. Recombinant dI and dIII (wild type and the E155W mutant) from R. rubrum transhydrogenase and human heart dIII were expressed in Escherichia coli and purified by column chromatography as described in Refs. 22Diggle C. Hutton M. Jones G.R. Thomas C.M. Jackson J.B. Eur. J. Biochem. 1995; 228: 719-726Crossref PubMed Scopus (59) Google Scholar, 23Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar, 24Peake S.J. Venning J.D. Cotton N.P.J. Jackson J.B. Biochim. Biophys. Acta. 1999; 1413: 81-91Crossref PubMed Scopus (18) Google Scholar, 25Peake S.J. Venning J.D. Jackson J.B. Biochim. Biophys. Acta. 1999; 1411: 159-169Crossref PubMed Scopus (26) Google Scholar. After supplementing with 25% glycerol, they were stored at –20 °C. Thawed proteins were either used directly in experiments or were first concentrated in Vivaspin centrifugal filters (5-kDa cut-off for dIII and 10-kDa cut-off for dI). Protein concentrations (given with respect to subunits) were determined by the microtannin procedure (26Mejbaum-Katzenellenb S. Drobryszycka W.J. Clin. Chem. Acta. 1959; 4: 515-522Crossref Scopus (156) Google Scholar). Complexes (dI2dIII1) of dI and dIII are generated spontaneously (Kd < 60 nm (27Venning J.D. Rodrigues D.J. Weston C.J. Cotton N.P.J. Quirk P.G. Errington N. Finet S. White S.A. Jackson J.B. J. Biol. Chem. 2001; 276: 30678-30685Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar)) upon mixing the two components in solution. The dIII proteins are normally isolated in their NADP+-bound forms. Where required, the NADP+ was replaced by NADPH as described in Ref. 15Venning J.D. Bizouarn T. Cotton N.P.J. Quirk P.G. Jackson J.B. Eur. J. Biochem. 1998; 257: 202-209Crossref PubMed Scopus (33) Google Scholar. To replace with thio-NADP+, human dIII and R. rubrum dIII were first washed in Vivaspin filters with 10 mm Tris-HCl, pH 8.0, 1 mm dithiothreitol, 4 μm NADP+. The protein (∼35 mg ml–1) was then incubated in 10 mm Tris-HCl, pH 8.0, 1 mm dithiothreitol, 10 mm thio-NADP+ at 4 °C for 1 h, a period sufficient to permit release of all tightly bound NADP+ (24Peake S.J. Venning J.D. Cotton N.P.J. Jackson J.B. Biochim. Biophys. Acta. 1999; 1413: 81-91Crossref PubMed Scopus (18) Google Scholar). In crystallization experiments and in measurements of the cyclic reaction (see below), this solution was used directly. In stopped flow experiments and in measurements of nucleotide release, the solution was washed again in 10 mm Tris-HCl, pH 8.0, 1 mm dithiothreitol, and 50 μm thio-NADP+. Everted cytoplasmic membranes (chromatophores) were isolated from phototrophically grown cultures of wild-type R. rubrum strain S1 and from a transhydrogenase-overexpressing strain RTB2 (28Bizouarn T. Sazanov L.A. Aubourg S. Jackson J.B. Biochim. Biophys. Acta. 1996; 1273: 4-12Crossref PubMed Scopus (34) Google Scholar) by French pressing the cells as described in Ref. 29Cunningham 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 bacteriochlorophyll concentration was determined using the in vivo extinction coefficient of 140 mm–1 cm–1 at 880 nm (30Clayton R.K. Biochim. Biophys. Acta. 1963; 73: 312-323Crossref Scopus (202) Google Scholar). Where indicated, the dI component was washed from the membranes by centrifugation in the absence of NADP(H) (22Diggle C. Hutton M. Jones G.R. Thomas C.M. Jackson J.B. Eur. J. Biochem. 1995; 228: 719-726Crossref PubMed Scopus (59) Google Scholar). Reconstitution with recombinant dI protein was achieved by simple mixing. Assays of steady state transhydrogenation were performed at 25 °C on a Perkin Elmer Lambda 16 double-beam spectrophotometer using extinction coefficients given in Refs. 21Rydstrom J. Methods Enzymol. 1979; 55: 261-275Crossref PubMed Scopus (37) Google Scholar and 31Venning J.D. Jackson J.B. Biochem. J. 1999; 341: 329-337Crossref PubMed Scopus (26) Google Scholar. The rate of the reverse reaction (compare Equation 1) with physiological nucleotides was determined in the presence of an NADPH-regenerating system comprising 6 μg ml–1 NADP-linked isocitrate dehydrogenase (Sigma I2002) and 4.0 mm isocitrate. Absorbance changes in the transient state were recorded at 20 °C using an Applied Photophysics DX17-MV in its absorbance mode; the mixing time of the instrument was 1.31 ms (15Venning J.D. Bizouarn T. Cotton N.P.J. Quirk P.G. Jackson J.B. Eur. J. Biochem. 1998; 257: 202-209Crossref PubMed Scopus (33) Google Scholar, 31Venning J.D. Jackson J.B. Biochem. J. 1999; 341: 329-337Crossref PubMed Scopus (26) Google Scholar). Protein fluorescence was measured using a Spex FluoroMax in the time drive mode. Heteronuclear single quantum coherence experiments (32Mori S. Abeyguanawardana C. Johnson M.O. Van Zijl P. J. Magn. Res. Series B. 1995; 108: 94-98Crossref PubMed Scopus (564) Google Scholar) on 15N-labeled protein were performed on a Bruker AMX500 NMR spectrometer, essentially as described in Ref. 33Quirk P.G. Jeeves M. Cotton N.P.J. Smith K.J. Jackson J.B. FEBS Lett. 1999; 446: 127-132Crossref PubMed Scopus (40) Google Scholar. Human dIII in its thio-NADP+ form was crystallized essentially as described for the protein in its NADP+ form (10White S.A. Peake S.J. McSweeney S. Leonard G. Cotton N.N.J. Jackson J.B. Structure. 2000; 8: 1-12Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Diffraction data were collected at beamline ID14–1 at the European Synchrotron Radiation Facility (Grenoble, France) using a MAR-CCD detector. The crystals were flash-cooled to 100 K in the cryostream immediately prior to data collection. A complete data set was collected to 2.4 Å. The R. rubrum dI2dIII1 complex in its thio-NAD+/NADP+ form was crystallized essentially as described previously for (dI.Q132N)2dIII1 mutant protein in its NAD+/NADP+ form (11Cotton N.P.J. White S.A. Peake S.J. McSweeney S. Jackson J.B. Structure. 2001; 9: 165-176Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 12van Boxel G.I. Quirk P. Cotton N.J.P. White S.A. Jackson J.B. Biochemistry. 2003; 42: 1217-1226Crossref PubMed Scopus (12) Google Scholar). A complete data set was collected to 2.6 Å on an ADSC detector on beamline ID 14–2 also at the European Synchrotron Radiation Facility. In both cases, the crystals were isomorphous with those loaded with the physiological substrates. The data were integrated and scaled using the programs MOSFLM (34Leslie A.G.W. Joint CCCP4 and ESF-EADBM Newsletter on Protein Crystallography. 1992; 226Google Scholar) and SCALA (35Evans P.R. Joint CCP4 and ESF-EACMB Newsletter. 1997; 33: 22-24Google Scholar). The wild-type structures of dIII with NADP+ bound and dI2dIII1 with NAD+/NADP+ bound were refined against the structure factor amplitudes of dIII with thio-NAD+ bound and dI2dIII1 with NAD+/NADP+ bound, respectively, using the program CNS (36Brunger A.T. Adams P.D. Clore G.M. Delano W.L. Gros P. Grossekunstleve R.W. Et A.L. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar, 37Grosse-Kunstleve R.W. Brunger A.T. Acta Crystallogr. Sect. D Biol. Crystallogr. 1999; 55: 1568-1577Crossref PubMed Scopus (52) Google Scholar). The refinement statistics are summarized in Table I. Simulated annealing omit maps, both f o – f c and 2f o – f c, calculated using CNS, confirmed the calculated positions of the atoms in the carbothiamide groups of the two structures. Further confirmation of the sulfur atom positions was obtained from test refinements using various combinations of the parameter files for the physiological and analogue nucleotides and the reflection files from the structures. The information for the sulfur atom positions was found to reside predominantly in the reflection files of the thio-NAD(P) files for both structures. The cut-off values for hydrogen bond determination were 2.35–3.2 Å. Ribbon diagrams were prepared using the programs MOLSCRIPT (38Kraulis P.J. J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and TURBO-FRODO (39Roussel A. Cambillau C. Silicon Graphics Geometry Partners Directory. Slicon Graphics, Mountain, CA1991: 86Google Scholar). The human dIII.thio-NADP+ structure and R. rubrum dI2dIII1 with bound thio-NAD+ and NADP+ appear as Protein Data Bank entries 1PT9 and 1PTJ, respectively.Table IData collection and refinement statistics for x-ray crystallographydI2dIII1·thioNAD+·NADP+ (R. rubrum)dIII·thio-NADP+ (human)Data collection statisticsaData in parentheses are for the highest resolution shell.Resolution (Å)42.26-2.61 (2.75-2.61)36.82-2.42 (2.55-2.42)Completeness (%)98.4 (98.4)98.5 (96.2)Multiplicity4.8 (4.8)6.0 (4.9)I/σI6.1 (2.0)6.8 (2.2)No. of observations162328 (23435)100817 (11365)No. of unique reflections33781 (4856)16825 (2312)R symbWhere R sym = Σj|〈I〉 — Ij |/Σ〈I〉 where Ij is the intensity of the jth reflection and 〈I〉 is the average intensity.6.1 (33.9)7.7 (31.9)Refinement statisticsWilson B/average B76.5/67.750.9/38.7No. of non-hydrogen atoms/waters6779/1462874/171r.m.s.d.cr.m.s.d., root mean square deviation from ideal values. bonds (Å)/angles (degree)0.0075/1.3870.0071/1.247RamachandrandThe values are percentages of amino acids in the “core,” “allowed,” “generously allowed,” and “disallowed” regions, respectively, according to the definition given in Ref. 53. (%)82.2, 16.1, 1.7, 0.089.8, 9.9, 0.3, 0.0R/R freee5% of data have been set aside for cross-validation calculations.23.52/28.7121.87/27.80a Data in parentheses are for the highest resolution shell.b Where R sym = Σj|〈I〉 — Ij |/Σ〈I〉 where Ij is the intensity of the jth reflection and 〈I〉 is the average intensity.c r.m.s.d., root mean square deviation from ideal values.d The values are percentages of amino acids in the “core,” “allowed,” “generously allowed,” and “disallowed” regions, respectively, according to the definition given in Ref. 53Laskowski R.A. Macarthur M.W. Moss D.S. Thornton J.M. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar.e 5% of data have been set aside for cross-validation calculations. Open table in a new tab Amino acid residues in R. rubrum dI and dIII are numbered according to their position in the recombinant proteins, as in Ref. 11Cotton N.P.J. White S.A. Peake S.J. McSweeney S. Jackson J.B. Structure. 2001; 9: 165-176Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar. Residues in the human dIII are numbered according to the sequence of the intact enzyme, as in Ref. 10White S.A. Peake S.J. McSweeney S. Leonard G. Cotton N.N.J. Jackson J.B. Structure. 2000; 8: 1-12Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar. The dihedral angle X am describes the twist of the carboxamide (or carbothiamide) group relative to the plane of the pyridine ring of a nicotinamide nucleotide. It is defined by viewing atoms C-2–C-3–C-7–O-7 (or S7) along C-3 → C-7; it is 0° when O-7 (or S7) is perfectly cis to C-2, positive for a clockwise rotation of the carboxamide (carbothiamide) from this value and negative for an anticlockwise rotation. Xn is the dihedral that describes the twist of the nicotinamide ring relative to the ribose ring across the glycosidic bond; it is defined by the atoms O-4–C-1–N-1–C-2 and is 0° when O-4 is cis to C-2. When Xn is between 0 and 180°, the rings are said to be in a syn conformation, and when Xn is between 0 and –180° they are anti. The dihedral angles were calculated using TURBO-FRODO. All of the nucleotides and nucleotide analogues were obtained from Sigma. Thio-NAD + Is a Poor Substrate in the Reverse and Cyclic Reactions Catalyzed by R. rubrum Transhydrogenase—The steady state rates of NADPH oxidation by NAD+, by thio-NAD+, and by AcPdAD+ (all “reverse” transhydrogenation reactions) in R. rubrum strain RTB2 membranes at saturating nucleotide concentrations, using the assay buffer described in the legend of Fig. 1, were 8.5, 4.5, and 16.0 mol mol–1 bacteriochlorophyll min–1, respectively. However, the rates of these reactions do not closely reflect events at the hydride transfer step because reverse transhydrogenation is at least partly limited by slow NADP+ release (40Bizouarn T. Stilwell S.N. Venning J.M. Cotton N.P.J. Jackson J.B. Biochim. Biophys. Acta. 1997; 1322: 19-32Crossref PubMed Scopus (29) Google Scholar). “Cyclic” transhydrogenation (Scheme 1) more reliably indicates the rate of hydride transfer because it can proceed without NADP+ (or NADPH) leaving the enzyme (41Hutton M.N. Day J.M. Bizouarn T. Jackson J.B. Eur. J. Biochem. 1994; 219: 1041-1051Crossref PubMed Scopus (79) Google Scholar). Fig. 1 shows that the maximum rate of cyclic reduction of thio-NAD+ by NADH plus NADPH (Scheme 1A) was only 5.5 mol mol–1 bacteriochlorophyll min–1, whereas the maximum rate of cyclic AcPdAD+ reduction by NADH plus NADPH (Scheme 1B) under equivalent conditions was 130 mol mol–1 bacteriochlorophyll min–1. The left-hand arms of these two cyclic reactions (that is, the reduction of bound NADP+ by NADH in Scheme 1, A and B) are identical. Therefore, the oxidation of bound NADPH by AcPdAD+ in intact transhydrogenase is much faster than that by thio-NAD+ (the right-hand arms). Despite the large difference in rate, the Km for thio-NAD+ is only about 2.5 times greater than that for AcPdAD+ in the respective reactions. We were unable to detect any oxidation of thio-NADH by NADP+ (a forward transhydrogenation) in R. rubrum membranes under either darkened or illuminated conditions.Scheme 1The cyclic reaction of transhydrogenase. E represents an enzyme catalytic site at the interface between dI and dIII. The dIII nucleotide-binding site is shown to be permanently occupied by either NADP+/NADPH (A and B) or thio-NADP+/thio-NADPH (C). The two double-headed arrows in each panel show consecutive events at the catalytic site. For example, in A, at the left arrows, NADH binds (to dI) and reduces the (dIII-bound) NADP+, and NAD+ then dissociates; at the right arrows, thio-NAD+ binds (to dI) and oxidizes the (dIII-bound) NADPH, and thio-NADH then dissociates.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mixtures of isolated, purified dI and dIII of R. rubrum transhydrogenase spontaneously form stable dI2dIII1 complexes (11Cotton N.P.J. White S.A. Peake S.J. McSweeney S. Jackson J.B. Structure. 2001; 9: 165-176Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 22Diggle C. Hutton M. Jones G.R. Thomas C.M. Jackson J.B. Eur. J. Biochem. 1995; 228: 719-726Crossref PubMed Scopus (59) Google Scholar, 27Venning J.D. Rodrigues D.J. Weston C.J. Cotton N.P.J. Quirk P.G. Errington N. Finet S. White S.A. Jackson J.B. J. Biol. Chem. 2001; 276: 30678-30685Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The steady state rates of oxidation of NADPH by NAD+, by thio-NAD+, and by AcPdAD+ catalyzed by dI2dIII1 complexes were all very similar (∼2 mol mol–1 dIII min–1) but, even more than in the intact enzyme, these reverse transhydrogenations are limited by the very low rate of product NADP+ release (23Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar). Cyclic reduction of thio-NAD+ by NADH plus NADPH catalyzed by dI2dIII1 complexes (140 mol mol–1 dIII min–1; Scheme 1A) was considerably slower than cyclic reduction of AcPdAD+ by NADH plus NADPH (typically 2000–3000 mol mol–1 dIII min–1 (12van Boxel G.I. Quirk P. Cotton N.J.P. White S.A. Jackson J.B. Biochemistry. 2003; 42: 1217-1226Crossref PubMed Scopus (12) Google Scholar, 23Diggle C. Bizouarn T. Cotton N.P.J. Jackson J.B. Eur. J. Biochem. 1996; 241: 162-170Crossref PubMed Scopus (60) Google Scholar); Scheme 1B). Following the same arguments as above, this indicates that, as in the intact enzyme, thio-NAD+ is a very poor acceptor of hydride equivalents from NADPH. Experiments in the stopped flow spectrophotometer provide complementary information. It was previously shown that mixing NADPH-loaded dI2dIII1 complexes with AcPdAD+ leads to a rapid burst of hydride transfer preceding the steady state reaction (15Venning J.D. Bizouarn T. Cotton N.P.J. Quirk P.G. Jackson J.B. Eur. J. Biochem. 1998; 257: 202-209Crossref PubMed Scopus (33) Google Scholar); the burst arises because the binding of AcPdAD+, hydride transfer, and release of AcPdADH are all very fast relative to the rate of NADP+ release. Subsequently, measurements of changes in Trp fluorescence revealed an equivalent rapid burst of reaction between NADPH-loaded dI2dIII1 complexes and NAD+ (42Pinheiro T.J.T. Venning J.D. Jackson J.B. J. Biol. Chem. 2001; 276: 44757-44761Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 43Venning J.D. Peake S.J. Quirk P.G. Jackson J.B. J. Biol. Chem. 2000; 275: 19490-19497Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). In the experiment shown in Fig. 2, NADPH-loaded dI2dIII1 complexes were mixed in the stopped flow spectrophotometer with thio-NAD+. A burst of reaction was observed but with a much slower rate than that observed with either NAD+ or AcPdAD+ as hydride acceptors. Approximately similar concentrations of AcPdAD+, NAD+, and thio-NAD+ were required to give the maximum rate constants for the respective reactions (k app was ∼550 s–1 for NADPH → AcPdAD+, ∼600 s–1 for NADPH → NAD+, and ∼8 s–1 for NADPH → thio-NAD+). In a subsequent experiment, dIII loaded with NADPH from one syringe was mixed with dI plus thio-NAD+ (1.0 mm after mixing) from the other. Again the burst kinetics were observed and with a k app = ∼6s–1 (data not shown), proving that the slow rate of reaction is not a result of slow binding of thio-NAD+ to dI. The analysis of the k app values in terms of their microscopic rate constants, for AcPdAD+ and NAD+ as hydride acceptors, was discussed previously (15Venning J.D. Bizouarn T. Cotton N.P.J. Quirk P.G. Jackson J.B. Eur. J. Biochem. 1998; 257: 202-209Crossref PubMed Scopus (33) Google Scholar). The transient state kinetics of forward transhydrogenation on dI2dIII1 complexes with AcPdADH/NADP+ and with NADH/NADP+ were described in earlier works (31Venning J.D. Jackson J.B. Biochem. J. 1999; 341: 329-337Crossref PubMed Scopus (26) Google Scholar, 42Pinheiro T.J.T. Venning J.D. Jackson J.B. J. Biol. Chem. 2001; 276: 44757-44761Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar, 43Venning J.D. Peake S.J. Quirk P.G. Jackson J.B. J. Biol. Chem. 2000; 275: 19490-19497Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar). These reactions also take place as a rapid single-turnover burst, here the slow steady state rate resulting from limiting NADPH release. For AcPdADH/NADP+, measured from the 375 nm absorbance change at saturating AcPdADH, k app = ∼90 s–1, and for NADH/NADP+, measured from a Trp fluorescence change under continuous flow conditions at saturating NADH, k app = ∼21000 s–1. When the dI2dIII1 complex loaded with NADP+ was mixed with thio-NADH in the stopped flow spectrophotometer, a single-turnover burst of reaction was observed, but it was considerably slower than with either AcPdADH or NADH as hydride donor (k
Referência(s)