Eur J Biochem 269, 4505–4515 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03144.x Redox-sensitive loops D and E regulate NADP(H) binding in domain III and domain I–domain III interactions in proton-translocating Escherichia coli transhydrogenase Carina Johansson1, Anders Pedersen1, B Goran Karlsson2 and Jan Rydstrom1 ă ă Department of Biochemistry, Goăteborg University, Sweden; 2Department of Molecular Biotechnology, Chalmers University of Technology, Goăteborg, Sweden Membrane-bound transhydrogenases are conformationally driven proton-pumps which couple an inward proton translocation to the reversible reduction of NADP+ by NADH (forward reaction) This reaction is stimulated by an electrochemical proton gradient, Dp, presumably through an increased release of NADPH The enzymes have three domains: domain II spans the membrane, while domain I and III are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively Separately expressed domain I and III together catalyze a very slow forward reaction due to tightly bound NADP(H) in domain III With the aim of examining the mechanistic role(s) of loop D and E in domain III and intact cysteine-free Escherichia coli transhydrogenase by cysteine mutagenesis, the conserved residues bA398, bS404, bI406, bG408, bM409 and bV411 in loop D, and residue bY431 in loop E were selected In addition, the previously made mutants bD392C and bT393C in loop D, and bG430C and bA432C in loop E, were included All loop D and E mutants, especially bI406C and bG430C, showed increased ratios between the rates of the forward and reverse reactions, thus approaching that of NADPH values the wild-type enzyme Determination of kd indicated that the former increase was due to a strongly increased dissociation of NADPH caused by an altered conformation of loops D and E In contrast, the cysteine-free G430C mutant of the intact enzyme showed the same inhibition of both forward and reverse rates Most domain III mutants also showed a decreased affinity for domain I The results support an important and regulatory role of loops D and E in the binding of NADP(H) as well as in the interaction between domain I and domain III Transhydrogenase is a membrane protein, which is found in the inner membrane of mitochondria and in the cytoplasmic membrane of bacteria It couples the reduction of NADP+ by NADH to the electrochemical proton gradient (Dp) according to the reaction Transhydrogenase from Escherichia coli is composed of an a subunit of about 54 kDa and a b subunit of about 48 kDa The active form of the enzyme is a2b2 Like all other membrane-bound transhydrogenases, the E coli enzyme is composed of three domains Domain I (dI) and domain III (dIII) are hydrophilic and contain the binding sites for NAD(H) and NADP(H), respectively, whereas domain II (dII) spans the membrane The genes encoding the hydrophilic and nucleotide-binding domains of transhydrogenase from several species have been overexpressed and the proteins have been purified and characterized In all cases, dI is purified as a dimer and lack bound substrates Separately expressed dIII exists as a monomer and contains tightly bound NADP(H), reflecting a dramatically increased affinity for NADP(H) as compared to the intact enzyme Even in the absence of the membrane bound dII, dI and dIII from the same or different species form a catalytically active complex capable of catalyzing the various transhydrogenation reactions However, the tight binding of NADP(H), results in low reactions rates for the reverse and forward reactions catalyzed by the dI + dIII complex, as these reactions are limited by a slow release of NADP+ and NADPH, respectively (reviewed in [2–4]) The 3D structures of dI from Rhodospirillum rubrum [5] and dIII from bovine [6], human [7], E coli [8–10] and R rubrum [11,12] transhydrogenases have been studied by both X-ray crystallography [5–7] and NMR [8–12] The global fold of dIII is a six-stranded parallel b sheet NADH ỵ NADPỵ ỵ Hỵ ! NADỵ ỵ NADPH ỵ Hỵ outị inị 1ị Out and in denote the cytosol and matrix, respectively, in mitochondria and periplasmic space and cytosol, respectively, in bacteria Key features of this reaction is that?p stimulates the rate of reduction of NADP+ by NADH some 10-fold and causes a shift in the apparent equilibrium constant from to approximately 500 [1] Correspondence to J Rydstrom, Department of Biochemistry, ă Goteborg University, Box462, 405 30 Goteborg, Sweden ă ă Fax: + 46 31 7733910, Tel.: + 46 31 7733921, E-mail: jan.rydstrom@bcbp.gu.se Abbreviations: dI, transhydrogenase domain I; dIII, transhydrogenase domain III; ecI, E coli dI; ecIII, E coli dIII; rrI, R rubrum dI; rrIII, R rubrum dIII; cfTH, cysteine-free transhydrogenase; AcPyAD+, oxidized 3-acetylpyridine-NAD+ MIANS, 2-(4¢-maleimidylanilino)naphthalene-6-sulfonic acid (sodium salt) (Received 30 May 2002, revised 12 July 2002, accepted 26 July 2002) Keywords: transhydrogenase; NADP; proton pump; membrane protein Ó FEBS 2002 4506 C Johansson et al (Eur J Biochem 269) surrounded by helices and irregular loops All dIII structures were solved using dIII with NADP+ bound in a nonclassical binding mode of the substrate, i.e as compared to other NADP(H)-dependent enzymes, NADP+ in dIII is bound in a reversed orientation The 3D structure of dIII with bound NADPH is still unknown, even though NMR experiments in which NADPH was added provided some information regarding regions that were affected by a change in the redox-state of the substrate [9–11] The crystal structure of dI from the R rubrum transhydrogenase [5] showed that it was a dimer, with the monomer comprised of the two subdomains A and 1B [5] The 1B subdomain has a Rossman fold responsible for binding NAD(H) in a novel mode as compared to other NAD(H)-binding enzymes [5] By using NMR in combination with mutagenesis, an extensive characterization of the dynamic interface between E coli transhydrogenase dI (ecI) and dIII (ecIII) was recently carried out [10] In addition to information regarding important residues involved in the ecI–ecIII interface, the results revealed unexpected redox-dependent changes of the ecI–ecIII interface suggested to be relevant for the overall reaction mechanism of the intact enzyme The regions at the C-terminal end of the b sheet comprised of residues bG389-bI406 (part of loop D, linking b-strands and 5) and bG430–bV434 (part of loop E, linking b strands and 6) were identified as redox-sensitive regions, regulated by the redox-state of NADP(H), that strongly influenced the ecI–ecIII interface [10] A structure of the dI2-dIII complex from R rubrum has recently been resolved by X-ray crystallography [13], which revealed dI–dIII interfaces at an atomic level similar to those derived from the NMR studies [10] Based on the crystal structures of dIII from the human heart [4,7] and the dI2-dIII complex from R rubrum [13], loops D and E have previously been suggested to be important in NADP(H)-binding and possibly also coupling to proton translocation [4,13] The corresponding regions in ecIII are indicated in an NMR-derived structural model (Fig 1) and in the amino-acid sequence with secondary structure elements (Fig 2) The functional importance of loops D and E have so far not been functionally established In the present work, the roles of these regions were investigated in greater detail using site-directed mutagenesis The results suggest that loop D is important in communicating affinity changes in the NADP(H)-binding site to domain I, and that both loop Fig The amino-acid sequence of ecIII and secondary structure elements based on NMR data Secondary elements were based on results from NMR experiments [10] Black shaded residues are conserved, and green-shaded residues are similar among different species of transhydrogenase D and E regulate the release of NADP(H) In support of the previous suggestion [4,13] both loops are suggested to play a key role in the regulation of the enzyme by an electrochemical proton gradient MATERIALS AND METHODS Site-directed mutagenesis The Quikchange mutagenesis kit (Stratagene) was used to introduce single cysteine mutations in isolated ecIII and cfTH A modified pET8c plasmid used for the preparation of histidine-tagged ecIII [14] served as a DNA template in the construction of the seven cysteine mutants ecIIIA398C, ecIIIS404C, ecIIII406C, ecIIIG408C, ecIIIM409C, ecIIIV411C and ecIIIY431C In addition, four previously produced mutants, i.e ecIIID392C [15], and ecIIIT393C, ecIIIG430C and ecIIIA432C [9], were further characterized The cfbG430C mutant was based on the pCLNH plasmid to which an N-terminal histidine tag has been added to the cfTH gene [14] The correctness of the mutant products was checked by DNA sequencing Fig Two views of the partial 3-dimensional structure of ecIII The NADP(H)-binding site of ecIII This illustration was based on the NMR structure of E coli dIII [10] The beginning and end of loop D (excluding a5) and E are P403-V411 and M427-G433, respectively The illustration was prepared using the software MOLMOL [26] Ó FEBS 2002 Redox-sensitive loops D and E in transhydrogenase (Eur J Biochem 269) 4507 Expression and purification Fluorescence measurements The gene encoding ecIII was based on the 177 C-terminal residues of E coli transhydrogenase b-subunit (bQ286bL462) EcIII mutants [14] and E coli cysteine-free transhydrogenase enzymes [15] were expressed and purified as described The plasmid pCD1 encoding R rubrum domain I (rrI) was expressed in E coli TG1 cells [16] and purified according to the method described by Bizouarn et al [17] with modifications After sonication and centrifugation of L culture, the supernatant was loaded onto a 15-mL Q-Sepharose HP column (Pharmacia) equilibrated with 20 mM Tris/HCl and 10 mM (NH4)2SO4 (pH 8.0) Protein was eluted with about 60 mL of the same buffer, after which (NH4)2SO4 was added to a final concentration of 1.6 M After 10 h of incubation the sample was centrifuged for h at 18 000 r.p.m in a Beckman JA20 rotor, and the supernatant loaded onto a 15-mL Butyl Toyopearl column (Tosohas) Protein was eluted with a gradient (300 mL) of 1.6 to M (NH4)2SO4 in 20 mM Tris/HCl (pH 8.0), dialyzed against 20 mM Tris/HCl, 10 mM (NH4)2SO4 pH 8.0 and stored at )20 °C in 20% glycerol All domains displayed a purity greater than 90% as judged by SDS-polyacrylamide gel electrophoresis using 8–25% gradient gels in the Phast system (Pharmacia) for transhydrogenase domains and 10–20% gradient gels (Novex) for the cfTH enzymes Fluorescence measurements were carried out on a SPEX Model FL1T1 s2 and Shimadzu RF5001PC spectrofluorometers at 25 °C A cuvette with a 10 · 10 mm cross section was used and the excitation and emission slits were both 2.5 nm Determination of the NADPH release rate from ecIII by fluorescence The release rate of NADPH from ecIII was determined from the exponential decrease in fluorescence as bound NADPH was released from ecIII and oxidized by glutathione and glutathione reductase using excitation and emission wavelengths of 340 and 460 nm, respectively Oxidized glutathione (2 mM) was added to the cuvette containing 1.5–2 lM of mutant ecIII enzyme and 1–4 U of glutathione reductase; the fluorescence was monitored for up to 20 In the case of the ecIIIV431C mutant, it was preincubated with an equimolar concentration (about lM) of NADPH for prior to the assay The measurements were carried out in a buffer composed of 20 mM Mops and mM MgCl2 (pH 7.0) RESULTS Characterization of single-cysteine mutations in ecIII Determination of protein concentration and substrate content Protein concentrations were determined using the bicinchoninic acid assay with bovine serum albumin as standard [18] The content of bound NADPH in the ecIII mutants was determined by absorbance spectroscopy at 339 nm, using eNADPH ¼ 6100 M)1Ỉcm)1, whereas the content of NADP+ was determined by fluorescence using a modified Klingenberg procedure as described previously [14] Activity assays Unless stated otherwise, transhydrogenation reactions catalyzed by mutant and wild-type ecIII were assayed as described [19] in buffer A [20 mM each of Mes, Mops, Ches, and Tris, 50 mM NaCl (pH 7.0)], using rrI Protein-protein titrations were performed in which the ecIII concentration was kept constant and the rrI concentration varied until a maximal rate was reached The forward and reverse reactions catalyzed by the cfTH and cfTH mutant enzymes were measured as described [20] in buffer B [20 mM each of Mes, Ches, Tris and Hepes, 50 mM NaCl and 0.01% Brij (pH 7.0)] whereas the cyclic reaction [19,20] was normally assayed in buffer C [20 mM each of Mes, Ches, Tris and Hepes, 50 mM NaCl, mM MgCl2, mM EDTA and 0.01% Brij (pH 6.0)] The reverse and cyclic reactions were followed optically at 375 nm as reduction of AcPyAD+ as described [19] The forward reaction was measured spectroscopically at 398 nm as reduction of thio-NADP+ [21] For comparative reasons, the transhydrogenation reactions catalyzed by rrI + wild-type and mutant ecIII mixtures were also assayed in the cfTH assay buffers All measurements were performed at 25 °C In order to elucidate the mechanistic roles of loop D and E in greater detail, the single ecIII cysteine mutants, ecIIIA398C, ecIIIS404C, ecIIII406C, ecIIIG408C, ecIIIM409C, ecIIIV411C (loop D), and ecIIIY431C (loop E), were expressed in the cysteine-free background and purified as described in Materials and methods In addition, the previously made mutants ecIIID392C and ecIIIT393C in loop D, and ecIIIG430C and ecIIIA432C in loop E, were included All mutants were characterized with respect to substrate binding, catalytic activities of the different transhydrogenation reactions and affinity for rrI In these assays and under the conditions used dimer formation by all mutants amounted to less than 10% as tested by SDS/ PAGE (data not shown) Figure A and B show the detailed positions of some of the mutated as well as other important residues in ecIII, and bound NADP+, in ecIII, viewed from two different angles Note that loops D and E are much more defined in the crystal structure of bovine dIII shown in Fig than in Fig A recently produced high resolution NMR structure of ecIII with bound NADP+ is essentially identical to that of bovine dIII with bound NADP+ (C Johansson, J T Johansson, A Pedersen, J Rydstrom and B G Karlsson, ă unpublished results) Content of bound NADP(H) in dIII The increased affinity of dIII for NADP(H) is reflected in the content of tightly bound NADP(H) in almost 100% of the molecules of separately expressed ecIII [10,19] The presence of NADP(H) changes the absorbance maximum, kmax, from 278 nm for the apo-protein to 268 nm for the NADP(H)-containing ecIII Consequently, kmax is an indication of the fraction of apo-protein An additional Ó FEBS 2002 4508 C Johansson et al (Eur J Biochem 269) Fig NADP(H) binding region of dIII viewed perpendicular to (A) and parallell with the b sheet (B) The structure was modelled using MOLMOL [26] based on the crystal structure of the bovine dIII with bound NADP+ (PDB entry 1D40) Important residues and loops D and E, are indicated Numbering of mutated residues is according to ecIII typical property of isolated ecIII is the percentage of bound NADP(H), which is highly reproducible for different preparations In order to assess the effects on substratebinding site occupancy, the proportions of bound NADP+ and NADPH were determined for the mutants generated in this investigation i.e ecIIIA398C, ecIIIS404C, ecIIII406C, ecIIIG408C, ecIIIM409C, ecIIIV411C and ecIIIY431C (Table 1) Table Content of bound NADP(H) in wild-type and mutant ecIII The concentrations of NADPH in the ecIII enzymes were calculated from UV-Vis spectra and the content of NADP+ was determined by a modified Klingenberg procedure (see Materials and methods) The kmax value corresponds to the wavelength at which maximal absorption was observed for the interval 200–400 nm in UV-Vis spectra Enzyme Loop affected kmax NADP+ (%) NADPH (%) Apo-form (%) ecIII ecIIIA398C ecIIIS404C ecIIII406C ecIIIG408C ecIIIM409C ecIIIV411C ecIIIY431C D D D D D D E 267 267 267 267 267 267 267 267 87 79 66 72 65 70 40 43 16 33 19 24 27 28 11 54 29 Ó FEBS 2002 Redox-sensitive loops D and E in transhydrogenase (Eur J Biochem 269) 4509 All ecIII mutants contained bound substrate, where the percentages of NADPH and NADP+ varied, often with an increased content of NADPH This effect was most pronounced in the case of the ecIIIS404C, ecIIIM409C and ecIIIY431C mutants, with 33%, 27% and 28% bound NADPH, respectively, and a relatively constant amount of apoprotein EcIIIV411C and ecIIIY431C did not follow the same pattern, but contained approximately 54% and 29% apoprotein, respectively (Table 1) Thus, it is clear that the mutations in the redox-sensitive loops D and E strongly affected the binding site In all mutants in loop D, except ecIIIV411C, the nucleotide content was at least 89%, with a 3–5 fold increase in the percentage of bound NADPH (Table 1) Clearly, the small fraction of apo-protein in these mutants reflected the fact that this region of the protein is not directly involved in substrate binding Forward reaction catalyzed by rrI + ecIII mutant mixtures The forward reaction (reduction of thio-NADP+ by NADH) catalyzed by rrI and ecIII mutant mixtures was examined by protein-protein titrations in which the ecIII concentration was kept constant and the rrI concentration varied until a maximal rate was reached In this and other dI-dependent assays in this investigation, rrI rather than ecI was used due to the generally higher activities obtained with this dI preparation (cf Fig 2) The [rrI]/[ecIII] ratio at halfsaturation is a measure of the affinity of the complex formed for rrI and ecIII [10] and may be used to gain information about the role of a particular amino-acid residue in the interactions with dI However, because the rrI concentration required for half maximal rate is dependent on both the Kd for the rrI + ecIII complex and the release rate of thioNADPH from domain III, it can not be considered as a true measure of the affinity between the two domains Due to the tight binding of nucleotides to wild-type ecIII, the forward reaction catalyzed by ecIII with saturating concentrations of rrI is limited at pH 7.0 by the slow release of thio-NADPH from domain III [16] An increase in the forward reaction rate may thus be regarded as an increase in the release rate of NADPH Table summarizes the results obtained for measurements of the forward reaction catalyzed by rrI and ecIII mutants The maximal rates for the ecIIIG430C and ecIIIY431C mutants were 275 and 100 times that of the wild-type ecIII, respectively This pronounced increase demonstrates the dramatic effect on the rate of dissociation of NADPH caused by mutations in loop E Depending on the position in which the cysteine residue was introduced, mutations in loop D had different consequences on the forward reaction rate The ecIIII406C mutant showed a 35-fold higher rate than wild-type ecIII, whereas the ecIIIS404C mutation had a minor effect (Table 2) Likewise, the ecIIIA398C, ecIIIG408C, ecIIIM409C, ecIIIV411C and ecIIIA432 mutants showed relatively minor changes As the ecIIII406 residue does not participate directly in substrate binding [6,7], the increased forward reaction rate demonstrated by the ecIIII406C mutant was possibly a result of perturbation of the surroundings of this position caused by the mutation An obvious candidate responsible for this effect is the conserved bD392 residue, Table Properties of the forward reaction catalyzed by wild-type and mutant ecIII in the presence of rrI The values are estimations from protein-protein titration curves in which the ecIII concentration was fixed and the rrI concentration varied (not shown) The assays were performed as described in Materials and methods The [ecIII] values refer to the fixed enzyme concentration used in the titrations The [rrI] values correspond to the concentration of rrI at half Vmax Vmax Enzyme [ecIII] (nM) [rrI] (nM) [rrI]/ [ecIII] (mol thio-NADPH)Ỉ (mol ecIII))1Ỉmin)1 % ecIII ecIIIA398C ecIIIS404C ecIIII406C ecIIIG408C ecIIIM409C ecIIIV411C ecIIIG430C ecIIIY431C ecIIIA432C 5000 2500 5000 2500 2500 5000 5000 2500 2500 2500 20 15 10 400 140 10 0.001 0.001 0.0008 0.08 0.002 0.003 0.002 0.16 0.06 0.004 0.04 0.12 0.08 1.4 0.13 0.13 0.22 11 0.13 100 300 200 3500 325 325 550 27500 10000 325 ˚ which is located within A from bI406 in the NADP+complexed dIII crystal structure [6,7] The [rrI]/[ecIII] at half-saturation was increased for all cysteine mutants, but appeared to be correlated to the maximal rate displayed by the mutants (Table 2) Reverse reaction catalyzed by rrI + ecIII mutant mixtures Analyses of the reverse reaction catalyzed by rrI + ecIII mutant mixtures were performed by protein-protein titrations in the same way as for the forward reaction Like the forward reaction, the reverse reaction is limited at pH 7.0 by the slow release of the product bound to dIII [16], but is several-fold faster The maximal rate of the reverse reaction is thus an excellent tool for examining if a mutation has altered the rate of dissociation of NADP+ The [rrI]/[ecIII] necessary for half Vmax is an indication of the dissociation constant for the rrI + ecIII complex but, like the forward reaction, this ratio is also dependent on how fast the product is released from ecIII An elevated release rate needs more dI to saturate the reaction As shown in Table the maximal rates obtained for both the ecIIII406C and ecIIIY431C mutants were 4.5-fold higher than that of wild-type ecIII, indicating an increased dissociation of NADP+ Again, the result obtained with the ecIIII406C mutant was probably an indirect effect caused by perturbations in its environment The ecIIIS404C, ecIIIG408C, ecIIIM409C and ecIIIV411C mutations did not affect the reverse reaction rate significantly, which is consistent with the fact that these positions are not in the substrate binding-site [6,7] The [rrI]/[ecIII] ratios at halfsaturation of the reverse reaction correlated well with the maximal rate of the ecIII mutants Cyclic reaction catalyzed by rrI + ecIII mutant mixtures The cyclic reaction, i.e the reduction of AcPyAD+ by NADH via NADP(H) bound to dIII, was analyzed by protein-protein titrations in which the ecIII concentration Ó FEBS 2002 4510 C Johansson et al (Eur J Biochem 269) Table Properties of the reverse reaction catalyzed by wild-type and mutant ecIII in the presence of rrI The values are estimations from protein-protein titration curves in which the ecIII concentration was fixed and the rrI concentration varied (not shown) The assays were performed as described in Materials and Methods The [ecIII] values refer to the fixed enzyme concentration used in the titrations The [rrI] values correspond to the concentration of rrI at half Vmax Vmax Enzyme [ecIII] (nM) [rrI] (nM) [rrI]/ [ecIII] (mol AcPyADH)Ỉ (mol ecIII))1Ỉmin)1 % ecIII ecIIIA398C ecIIIS404C ecIIII406C ecIIIG408C ecIIIM409C ecIIIV411C ecIIIY431C 4900 2500 4000 2500 2500 5000 2500 2500 20 30 30 250 40 150 110 240 0.004 0.012 0.008 0.100 0.016 0.030 0.044 0.096 4 18 18 100 100 75 450 100 75 125 450 was kept constant and the rrI concentration varied until the reaction rate reached a maximum NADP(H) remains bound to dIII during the entire catalytic cycle and the rate of the cyclic reaction at pH 7.0 has been shown to be limited by the hydride transfer steps [16] Measurements of the cyclic reaction thus offers an opportunity to examine the affinity of ecIII mutants for dI The [rrI][/ecIII] ratio at halfsaturation is only dependent on the dissociation constant for the rrI + ecIII complex Using the actual rrI and ecIII concentrations at half Vmax the Kd for the complex can be calculated according to Kd ¼ [rrI]-[ecIII]/2 In Table the data from the protein-protein titrations for the ecIII mutants are listed Except for the ecIIIS404C mutant, the rrI concentration required for half Vmax was considerably higher in all of the mutants made in the redox-sensitive loops The most affected mutants were the ecIIIA398C (loop D), ecIIIM409C (loop D) and ecIIIY431C (loop E) mutants which exhibited 6–15 times higher Kd for the rrI + ecIII complex as compared to wildtype ecIII (Table 4) In agreement with the crystal structure of the dI2–dIII complex [13], these results show that both loops D and E directly or indirectly make crucial contacts with dI Mutations in the I406–V411 region affected the hydride transfer efficiency and only 24–31% of the maximal rate of the cyclic reaction could be reached, despite saturating concentrations of rrI Interestingly, the ecIIIS404C and ecIIIA398C mutants were still able to catalyze the hydride transfer at a wild-type rate, even though the affinity for rrI had been substantially lowered Release rate of NADPH measured by fluorescence The fact that NADPH, but not NADP+, fluoresces at 460 nm when using excitation at 340 nm, was utilized in order to determine the rate of release of NADPH from ecIII By this method bound NADPH was oxidized by glutathione reductase and glutathione The reaction is limited by the rate of release of NADPH and the resulting decrease in fluorescence could consequently be used to calculate the KoffNADPH [22] Fig shows the oxidation by glutathione and glutathione reductase of NADPH bound to ecIIIG432C and ecIIIG430C The rate obtained with ecIIIA432C is representative of the wild-type rate In contrast, the ecIIIG430C mutant showed a dramatic 110fold increase in oxidation rate Based on similar oxidation traces, KoffNADPH values for several ecIII mutants were calculated (Table 5) The release rate of NADPH was only significantly increased for the ecIIII406C, ecIIIG430C and ecIIIY431C mutants In contrast to other mutants, the NADP(H) bound to ecIIIY431C was rapidly lost upon storage The NADPH released was subsequently oxidized, requiring reloading with equimolar NADPH prior to assay The ecIIII406C and ecIIIY431C mutants displayed a fourfold to fivefold faster release of NADPH These results also indicate that there is no obvious relationship between the release rate of NADPH and the content of bound NADP(H) (cf Table 1) Characterization of the G430C mutant in intact cysteine-free E coli transhydrogenase In order to examine the effects of a mutation in the redoxsensitive region of loop E in the intact E coli transhydrogenase, the cfTHG430C mutant was constructed and the Table Properties of the cyclic reaction catalyzed by wild-type and mutant ecIII in the presence of rrI The values are estimations from proteinprotein titration curves in which the ecIII concentration was fixed and the rrI concentration varied (not shown) The assays were performed as described in Materials and Methods The [ecIII] values refer to the fixed enzyme concentration used in the titrations The [rrI] values correspond to the concentration of rrI at 1/2 Vmax Kd is the estimated dissociation constant derived according to Kd ¼ [rrI]-[ecIII]/2 Vmax Enzyme [ecIII] (nM) [rrI] (nM) Kd (nM) (mol AcPyADH)Ỉ (mol ecIII))1Ỉmin)1 % ecIII ecIIIA398C ecIIIS404C ecIIII406C ecIIIG408C ecIIIM409C ecIIIV411C ecIIIY431C 40 12.5 12.5 40 40 40 40 40 70 300 95 250 200 450 150 470 50 294 89 230 180 430 130 450 4900 5000 4900 1400 1400 1200 1500 800 100 102 100 29 29 24 31 16 Ĩ FEBS 2002 100 % Ỉmin)1 100 175 13.4 0.9 )1 (lmol AcPyADH)Ỉ(mg cfTH) % 3.6 0.3 67 47 cfTH 0.5 cfTHG430C 0.05 )1 Ỉmin)1 100 10 (lmol AcPyADH)Ỉ(mg cfTH) )1 Ỉmin)1 % NADPH Km (lM) (lmol thio-NADPH)Ỉ(mg cfTH) resulting mutant protein was characterized with respect to catalytic activities The kinetic properties of the various transhydrogenation activities catalyzed by the cfTHG430C mutant are summarized in Table The severe effect of mutating this conserved glycine into a cysteine was clearly reflected in the resulting maximal rates of the reverse, forward and cyclic reactions which were all between and 10% of the corresponding wild-type cfTH activities The Km for NADPH in the reverse reaction was 40 times higher than that for wild-type cfTH whereas the Km for thioNADP+ in the forward reaction, remained essentially unchanged Consequently, the cfTHG430C mutation resulted in a substantial loss of affinity for NADPH, while the affinity for NADP+ was unaffected (Table 6) For comparative reasons, the transhydrogenation reactions catalyzed by rrI + wild-type ecIII and ecIIIG430C enzymes mixtures and by cfTH and cfTHG430C enzymes, Enzyme 0.4 0.8 110 Vmax 0.005 0.010 0.002 0.020 0.004 0.560 0.025 0.011 Vmax ecIII ecIIIT393C ecIIIS404C ecIIII406C ecIIIM409C ecIIIG430C ecIIIY431C ecIIIA432C thioNADPỵ Km (lM) Relative rate Vmax KoffNADPH (s)1) Cyclic reaction Enzyme Reverse reaction Table Release rates of NADPH from wild-type and mutant ecIII determined by fluorescence The release rates of NADPH from ecIII enzymes were determined by fluorescence as described in Materials and Methods The KoffNADPH was derived from curves obtained by monitoring the decrease in fluorescence as NADPH was oxidized Forward reaction Fig Release of NADPH from ecIII mutants studied by fluorescence KoffNADPH for ecIII mutants were estimated from curves obtained when monitoring the decrease in fluorescence intensity as ecIII enzymes were treated with glutathione and glutathione reductase (see Materials and methods) Upper trace denotes ecIIIA432C and lower trace ecIIIG430C thioNADPỵ NADPH Table Kinetic parameters of puried cfTH and cfTHG430C enzymes The Km , Km and Vmax values were derived from Eadie-Hofstee plots The fixed concentration of AcPyAD+ used in the reverse reaction was 400 lM for cfTH and 1500 lM for cfTHG430C The fixed concentration of NADH used in the forward reaction was 400 lM for cfTH and 800 lM for cfTHG430C For the cyclic reaction the following concentrations were used for cfTH; 200 lM NADP+, 200 lM AcPyaD+ and 100 lM NADH and for cfTHG430C; 1500 lM NADP+, 1500 lM AcPyAD+ and 750 lM NADH Redox-sensitive loops D and E in transhydrogenase (Eur J Biochem 269) 4511 Ó FEBS 2002 7.0 7.0 6.0 6.0 pH Æmin)1 7.0 7.0 7.0 5.0 2800 850 1370 87 )1 Vmax (molAcPyADH)Ỉ(mol ecIII) 0.04 48 rrI + ecIII rrI + ecIIIG430C cfTH cfTHG430C 7.0 7.0 7.0 7.0 32 370 31 Vmax (mol AcPyADH)Ỉ(mol ecIII) )1 Ỉmin)1 pH Vmax (mol thio-NADPH) (mol ecIII) )1 Ỉmin)1 pH Cyclic reaction Enzyme Conformational changes involved in the proton pumping mechanism of transhydrogenase have been suggested to be dependent on binding/release of NADP(H) and the redoxstate of this substrate [2,4] The exact location of NADP+ and the bonds stabilizing its association with dIII have been established both in isolated dIII [6,7] and in the dI2-dIII complex [13]; the two forms of dIII not reveal any major differences with regard to NADP+-binding [13] Structurally, loop E is involved in the binding of the pyrophosphate group as well as the ribose of 2¢-5¢-ADP through the conserved bG430 The preceding conserved K424-R425S426 residues bind the 2¢-phosphate [6,7,13] The structural role of loop D is less obvious, but it appears to interact with loop E as well as dI [13], and the semiconserved I406 points into an apparent crevice formed by loop D and E towards the nicotinamide ring of NADP+ The distance between ˚ I406 and the nicotinamide ring is about A [13] Based on this structural information, it was proposed that loop D and E (the ÔlidÕ) were involved in the interaction with dI and binding of NADP+, respectively, especially the change from the ÔoccludedÕ state to the open state and vice versa [2,4,13] These conclusions were supported by NMR studies where NADP+ had been replaced by NADPH in the R rubrum dIII [11] Studies of ecIII by NMR [10], especially chemical shifts caused by the presence of ecI and/or NADP(H), showed that the ecIII itself and the ecI–ecIII interface were altered upon a change in the redox-state of the bound NADP(H) Despite the large amount of structural information available for loops D and E, their functional roles have not been systematically examined by site-directed mutagegenesis Residues subjected to mutagenesis were therefore chosen based on the magnitude of their chemical shift perturbations in the NMR experiments [10], i.e especially G389-I406 and G430-V434, and surrounding residues, and on their conservation among 62 transhydrogenase gene sequences Some of these mutants, i.e ecIIIK424C, ecIIIH345C, ecIIIA348C, ecIIIR350C [15], and ecIIIT393C, ecIIIR425C, ecIIIG430C and ecIIIA432C [19], were partly characterized previously In order to be able to react these with thiol-specific reagents, all selected residues were mutated to cysteines in the cysteine-free E coli transhydrogenase Reverse reaction DISCUSSION Forward reaction were all analyzed in buffer C The maximal rates listed in Table were obtained from the pH optima of the respective mixtures of domains or enzymes For the reconstituted system, rrI + ecIII, there was a pronounced difference between the rates of the forward and reverse reaction, the reverse rate being 150 times higher For the reconstituted rrI + ecIIIG430C mutant, this difference in rates had largely disappeared, the reverse reaction being only 4.5 times faster (Table 7) Thus, the ratio between the forward and the reverse reaction rates was approximately the same for wild-type cfTH and cfTHG430C enzymes, but the activities displayed by the cfTHG430C mutant were only 8–10% of those catalyzed by wild-type cfTH (Table 7) However, the maximal rates of the forward and reverse reactions exhibited by the rrI + ecIIIG430C complex and the cfTHG430C enzyme were almost the same (Table 7) Table Comparison of the maximal rates of transhydrogenation reactions catalyzed by wild-type and G430C mutants The Vmax values listed in the table are the maximal rates obtained in the pH range 5.0–9.0 and are given with reference to mol ecIII The concentrations of substrates and enzymes were as described in the respective section 4512 C Johansson et al (Eur J Biochem 269) Ó FEBS 2002 Redox-sensitive loops D and E in transhydrogenase (Eur J Biochem 269) 4513 The role of loop D Mutations were introduced in loop D in ecIII, a region that was suggested by NMR experiments to be involved in redox-regulation of the interactions of ecIII with ecI [10] In addition to ecIIIA398C, ecIIIS404C and ecIIII406C, mutations were also made in the adjacent G408-M409-P410-V/ I411 region, i.e ecIIIG408C, ecIIIM409C and ecIIIV411C Earler made mutants in this region include ecIIID392C [15] and ecIIIT393 [10] All of these mutants show a varying content of bound NADP(H), the most conspicuous being ecIIIR425C [10] and ecIIID392C [15] which are isolated as 100% apo-form, and ecIIIV411C which has 54% apo-form (Table 1) Introduction of a cysteine in the S404 position of ecIII had little or no effect on the substrate-binding properties and affinity for dI This ecIII mutant behaved wild-type like in all experiments It should be noted, however, that the replacement of serine with cysteine is a rather mild substitution When the ecIIIS404C mutant was reacted with MIANS and NEM (A Pedersen, C Johansson and J Rydstrom, unpublished results), the reverse reaction was ¨ stimulated by a factor of 1.6 and 2.0, respectively, indicating that the side-chain of S404 is pointing towards the substrate The ecIIII406C mutation led to higher release rates for NADP+ and especially NADPH, as indicated by elevated reverse and forward reaction rates, as well as a faster release of NADPH in fluorescence measurements These observations might seem peculiar as the I406 residue does not make any specific interactions with NADP(H) in the crystal structure [6,7,13] However, as a working hypothesis, it is possible that I406 creates a suitable environment for the essential substrate-binding D392 This aspartic acid residue ˚ is located within A from I406 in the crystal structure and has been proposed on the basis of mutagenesis to be a key residue in catalysis/binding as well as the proton pumping, possibly constituting one end of the proton wire [2,10,15,20,23–25,27] Structural evidence [2,4,6,7] supports these proposals I406 may take part in the regulation of the accessibility of the D392 side-chain and thereby control protonation events The rrI affinity was also affected by the ecIIII406C mutation, as shown by the five-fold increase in the Kd for the rrI–ecIII complex The local environment of I406C was recently demonstrated by MIANS labeling experiments to depend on the redox-state of the added substrate (A Pedersen, C Johansson, B.G Karlsson & J Rydstrom, unpublished, ă results) This difference might reect a movement of the I406C side-chain, and probably the entire loop D, that is coupled to events in the NADP(H)-binding site Mutations in the G408-M409-P410-V/I411 region did not influence the substrate-binding characteristics of ecIII, but caused a substantial decrease in the affinity for domain I The Kd for the rrI + ecIIIM409C complex was ninefold higher than that for wild-type ecIII and the maximal rate of the cyclic reaction was only about 24%, indicating that the complex was distorted by this mutation The role of loop E Mutations in loop E in isolated ecIII had dramatic consequences on its interactions with both dI and NADP(H) The most remarkable property of these mutants was the high dissociation rates of NADP(H), suggested by both high forward (Table 2) and reverse (Table 3) reaction rates, particularly the fast release of NADPH in fluorescence measurements The ecIIIG430C and ecIIIA432C mutants were earlier shown to be catalyze 850- and 150-fold increased reverse rates, respectively [10] In the presence of rrI, the forward reaction catalyzed by the ecIIIG430C and ecIIIY431C mutants was 275 and 100 times faster, respectively, than that of wild-type ecIII As this reaction catalyzed by the rrI + ecIII complex normally is limited by the slow release of the NADPH, these high rates indicate high dissociation rates of NADPH Indeed, measured directly, a 110-fold increase in the KoffNADPH was demonstrated for the ecIIIG430C mutant A careful analysis of the structure of ecIII revealed that a plausible explanation for the above observation is that the side-chains of G430-Y431-A432 appear to be involved in specific interactions with NADP(H) and thus could contribute to an increased affinity for this substrate (see [6,7,13]) The side-chain of a cysteine residue most likely adopts a different angle than those of the original GYA residues This is particularly apparent from the content of bound NADP(H) and percentage apo-form of the ecIII mutants In contrast to wild-type ecIII, both ecIIIG430C [10] and ecIIIA432C [10] have a reversed content of NADP(H), i.e predominantly NADPH with no or little NADP+ cIIIY431C also has an altered NADP(H) content, and all three mutants have high apo-form content In addition to being important for the regulation of NADPH release, loop E also plays a crucial role in the interactions with dI This was demonstrated by the high concentration of rrI needed for half-saturation of the cyclic reaction catalyzed by the ecIIIY431C mutant (Table 4), as well as the ecIIIG430C and ecIIIA432C mutants [10] The Kd for the rrI + ecIIIG430C and rrI + ecIIIG430C complex was about 10 and 15 times, respectively, higher than that for the rrI + ecIII complex As expected, the rate of the cyclic reaction was inversely proportionate to the Kd value (Table and [10]), whereas the rate of the reverse reaction catalyzed by the ecIIIG430C [10], ecIII/431C (Table 3) and ecIIIA432C [10] mutants was proportionate to the [rrI]/[ecIII] ratio at 1/2 Vmax, respectively The inherent tight binding of NADP(H) in isolated domain III has previously been explained by the hypothesis that separately expressed dIII mimics the ‘occluded’ conformation of domain III in the intact enzyme [2,4,16] This occluded conformation is assumed to correspond to a state in which the hydride transfer step takes place [2] Consequently, the activities of the reverse and forward reactions catalyzed by rrI + ecIII, in which release of NADP(H) is limiting, were very low as compared to that of intact cfTH (Table 7) The comparison of the rates of the various transhydrogenation reactions catalyzed by wild-type and mutants of rrI + ecIII complexes and intact transhydrogenases allowed an important conclusion regarding the differences between isolated ecIII and dIII as it functions in intact transhydrogenase The ratio of the rates of the reverse and forward reactions catalyzed by rrI + ecIIIG430C was similar to that for cfTHG430C, i.e approximately (Table 6) Normally, this ratio is the same for cfTH, but 150 for rrI + ecIII (Table 6) Introduction of a cysteine residue in the G430 position 4514 C Johansson et al (Eur J Biochem 269) of ecIII obviously perturbed the conformation of loop E in such a way that allowed it to function essentially as in the wild-type intact enzyme/cfTH, i.e with a strongly increased rate of the forward reaction and a less strong increase in the reverse reaction Indeed, this change is consistent with the 40-fold elevated KmNADPH for cfTHG430C while Kmthio–NADP+ was unchanged The elevated Km for NADPH is most likely a consequence of a higher dissociation rate of this substrate, assuming that the Kon of NADPH is unchanged Thus, even though the evidence is rather indirect, it is conceivable that the tight binding of NADP(H) in the occluded state of domain III directly or indirectly involves G430 and/or the conformation of loop E An interesting possibility is therefore that G430 and loop E in the resting state of the intact enzyme (and in the ecIIIG430C mutant) are much less associated with NADP(H), i.e the ÔlidÕ is open As proton translocation in transhydrogenase is very likely associated with conformational changes that affect binding and release of NADP(H) [2,4], loop E may play a major role in the coupling mechanism of transhydrogenase In addition, the changes in affinity for NADP(H) could be communicated to domain I as loop E forms part of the region that confers a redox regulation of the ecI + ecIII complex interface In conclusion, the present results suggest that loop D is involved in the interactions with domain I and that the I406 residue is a potential candidate for the regulation of the accessibility of the side-chain of the D392 residue that is essential for proton-pumping Moreover, the results support the notion that loop E functions as a mobile lid [4,7,13], regulating the release of NADPH, a step that probably is of central importance in the coupling mechanism of transhydrogenase It is proposed that movements of these two loops work in concert to regulate the affinity of NADP(H), protonation events in their surroundings and to communicate these changes to domain I ACKNOWLEDGEMENTS This work was supported by the Swedish Natural Science Research 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beta K424 mutants Biochemistry 38, 1652–1658 25 Bragg, P.D & Hou, C (2001) Characterization of mutants of b aspartate213, and b aspartate222, possibly components of the energy transduction pathway of the proton-translocating pyridine nucleotide transhydrogenase of Escherichia coli Arch Biochem Biophys 388, 299–307 26 Koradi, R., Billeter, M & Wuthrich, K (1996) MOLMOL: ă a program for display and analysis of macromolecular structures J Mol Graphics 14 (51–5), 29–32 27 Rodrigues, D.J., Venning, J.D., Quirk, P.G & Jackson, J.B (2001) A change in ionization of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase regulates both hydride transfer and nucleotide release Eur J Biochem 268, 1430–1438  ... rates of NADPH from wild-type and mutant ecIII determined by fluorescence The release rates of NADPH from ecIII enzymes were determined by fluorescence as described in Materials and Methods The... purified as described in Materials and methods In addition, the previously made mutants ecIIID392C and ecIIIT393C in loop D, and ecIIIG430C and ecIIIA432C in loop E, were included All mutants were... (2000) Interactions of the NADP(H)- binding domain III of proton-translocating transhydrogenase from Escherichia coli with NADP (H) and the NAD (H) -binding domain I studied by NMR and site-directed