The relation between the soluble factor associated with H+-transhydrogenase of Rhodospirillum rubrum and the enzyme from mitochondria and Escherichia coli

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to proton translocation across the membranes of bacteria and mitochondria. The protein has a tridomain structure. Domains I and III protrude from the membrane (e.g. on the cytoplasmic side in bacteria) and domain II spans the membrane. Domain I has the binding site for NAD ϩ /NADH, and domain III for NADP ϩ /NADPH. We have separately purified recombinant forms of domains I and III from Rhodospirillum rubrum transhydrogenase. When the two recombinant proteins were mixed with substrates in the stopped-flow spectrophotometer, there was a biphasic burst of hydride transfer from NADPH to the NAD ϩ analogue, acetylpyridine adenine dinucleotide (AcPdAD ϩ ). The burst, corresponding to a single turnover of domain III, precedes the onset of steady state, which is limited by very slow release of product NADP ϩ (kϷ0.03 s Ϫ1 ). Phase A of the burst (kϷ600 s Ϫ1 ) probably arises from fast hydride transfer in complexes of domains I and III. Phase B (kϷ10Ϫ50 s Ϫ1 ), which predominates when the concentration of domain I is less than that of domain III, probably results from dissociation of the domain I:III complexes and further association and turnover of domain I. Phases A and B were only weakly dependent on pH, and it is therefore unlikely that either the hydride transfer reaction, or conformational changes accompanying dissociation of the I:III complex, are directly coupled to proton binding or Keywords : transhydrogenase; stopped flow; proton translocation; recombinant protein; membrane protein.Transhydrogenase couples the transfer of reducing equivalents (hydride ion equivalents) between NAD(H) and NADP(H) to the translocation of protons across a membrane (for reviews,(1) The enzyme is found in animal mitochondria and in bacteria. Probably, under most physiological conditions, the reaction is driven to the right, towards NADPH formation, by the proton motive force generated by the respiratory (or photosynthetic) electron-transport chain.Transhydrogenase has three domains. Domains I and III protrude from the membrane and possess the nucleotide-binding sites ; domain I for NAD ϩ and NADH, and domain III for NADP ϩ and NADPH [1Ϫ3]. Domain II spans the membrane and might comprise 10Ϫ12 transmembrane helices [4].Recombinant forms of domain I from Rhodospirillum rubrum [5,6] and Escherichia coli [7,8] and purified. The isolated domains bind their respective nucleotides with high specificity and affinity [5Ϫ10, 11]. A mixture of the two domains from the R. rubrum protein catalyses transhydrogenation, even in the absence of the membrane-spanning domain II [6,9], but the rates of the 'forward' and 'reverse' transhydrogenation reactions catalysed by the mixture are very slow Ϫ they are profoundly limited by release of product NADPH (k off Ϸ 5ϫ10 Ϫ4 s Ϫ1 ) and NADP ϩ (k off Ϸ 0.03 s Ϫ1 ), respectively, from domain III [9]. Evidently, in the complete enzyme, release of NADP(H) is accelerated by domain II. The mixture of domains I and III...

Section: Discussionmentioning

confidence: 99%

Stopped‐flow kinetics of hydride transfer between nucleotides by recombinant domains of proton‐translocating transhydrogenase

Venning¹,

Bizouarn²,

Cotton³

et al. 1998

“…Membranes of R. rubrum were washed with buffer in the absence of NADP' to remove native Th, [15]. Th, and depleted membranes were reconstituted and assayed by recording the rate of reduction of AcPdAD' by NADPH at 375 nm in the dark [26], or the reduction of [S]NADP+ by NADH at 395 nm during photosynthetic illumination [27].…”

Section: R Rubrummentioning

confidence: 99%

“…The genes for transhydrogenase from R. rubrum were cloned and sequenced by us [ 11,241 and, independently, using our peptide sequences [15], by Yamaguchi and Hatefi [12]. We have expressed the gene for Th, at high levels in Escherichia coli and shown that the resulting protein is dimeric and is active in reconstitution [25].…”

mentioning

confidence: 99%

Conformational Dynamics of a Mobile Loop in the NAD(H)‐Binding Subunit of Proton‐Translocating Transhydrogenases from Rhodospirillum Rubrum and Escherichia Coli

Diggle¹,

Cotton²,

Grimley³

et al. 1995

Self Cite

Transhydrogenase catalyses the reversible transfer of reducing equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane. Uniquely in Rhodospirillum rubrum, the NAD(H)-binding subunit (called Th,) exists as a separate subunit which can be reversibly dissociated from the membrane-located subunits. We have expressed the gene for R. rubrum Th, in Escherichia coli to yield large quantities of protein. Low concentrations of either trypsin or endoproteinase Lys-C lead to cleavage of purified Th, specifically at Lys227-Thr228 and Lys237-Glu238. Observations on the onedimensional 'H-NMR spectra of Th, before and after proteolysis indicate that the segment which straddles the cleavage sites forms a mobile loop protruding from the surface of the protein. Alanine dehydrogenase, which is very similar in sequence to the NAD(H)-binding subunit of transhydrogenase, lacks this segment. Limited proteolytic cleavage has little effect on some of the structural characteristics of Th, (its dimeric nature, its ability to bind to the membrane-located subunits of transhydrogenase, and the short-wavelength fluorescence emission of a unique Trp residue) but does decrease the NADH-binding affinity, and does lower the catalytic activity of the reconstituted complex. The presence of NADH protects against trypsin or Lys-C cleavage, and leads to broadening, and in some cases, shifting, of NMR spectral signals associated with amino acid residues in the surface loop. This indicates that the loop becomes less mobile after nucleotide binding. Observation by NMR during a titration of Th, with NAD' provides evidence of a two-step nucleotide binding reaction. By introducing an appropriate stop codon into the gene coding for the polypeptide of E. coli transhydrogenase cloned into an expression vector, we have prepared the NAD(H)-binding domain equivalent to Th,. The E. coli protein is sensitive to proteolysis by either trypsin or Lys-C in the mobile loop. Judging by the effect of NADH on its NMR spectrum and on the fluorescence of its Trp residues, the protein is capable of binding the nucleotide though it is unable to dock with the membrane-located subunits of transhydrogenase from R. rubrum.

“…rubrum as described [18]. The soluble transhydrogenase component (Th,) was purified as described [ 181 then dialysed against 1 .O M KCI, 1 mM MgCI,, 40 pM NADP' to lower the buffer concentration.…”

Section: Methodsmentioning

confidence: 99%

“…rubrum. Thus, removal of Th, (the 43-kDa water-soluble, NAD'/NADH binding domain of H'-Thase [18]) from the chromatophores, inactivated transhydrogenation activity in parallel with transhydrogenase-driven alkalinisation of the medium. Reconstitution with pure Th, led to parallel recovery of these activities and an H'/H-ratio that was similar to that in the native chromatophores ( Table 1).…”

Section: The H+/w Ratio Of Transhydrogenasementioning

confidence: 99%

The ratio of protons translocated/hydride ion equivalent transferred by nicotinamide nucleotide transhydrogenase in chromatophores from Rhodospirillum rubrum

Bizouarn

Jackson

1993

The reduction of acetylpyridine adenine dinucleotide (AcPdAD', an NAD' analogue) by NADPH, in chromatophores treated with valinomycin, was accompanied by alkalinisation of the external medium, as measured by the absorbance change of added cresol red, a simple, non-binding pH indicator. Experiments with a stopped-flow spectrophotometer showed that initial (linear) rates of alkalinisation persisted for 1-2s. From the results of experiments in which H' uptake was driven by a series of short flashes of light, the dependence of the outward proton leak on the extent of H ' uptake was established. Thus, the proton leak was subtracted from the initial rate of alkalinisation during transhydrogenation to give the true proton-uptake rate. The correction factor was usually about 10%. The ratio of protons translocated/H-transferred from NADPH to AcPdAD' (the H' / H-ratio) was 0.60 2 0.06.The transhydrogenation reaction between NAD ' and NADPH was measured in the presence of a regeneration system for NAD' (pyruvate and lactate dehydrogenase). In addition to the accompanying proton-translocation reaction, scalar H' consumption linked to the regeneration system was observed and permitted internal checks on the calibration of the cresol red absorbance changes. After correction for the proton leak and scalar proton uptake, an H + / K ratio of 0.60 2 0.30 was calculated from the initial rates.The water-soluble polypeptide of transhydrogenase (Th,) was washed from a sample of chroinatophores to inhibit transhydrogenation activity and the accompanying H + uptake. Re-addition of purified Th, to depleted chromatophores led to recovery of transhydrogenation activity and of H i uptake. In this reconstituted system the H'/H-was similar to that in the native membranes. These results make it unlikely that the H'/H-ratio is artefactually low because chromatophores have a population of transhydrogenase which is not coupled to proton translocation. Further evidence that the mechanistic H'/H-ratio of chromatophore transhydrogenase is less than 1 was provided by an analysis of the kinetics of alkalinisation of the medium during reduction of AcPdAD' by NADPH. It was shown that the progress of the transhydrogenation-induced alkalinisation was fitted by the sum of H' uptake (the rate of transhydrogenation multiplied by the H '/Hratio) plus the H' leak, when the ratio was 0.6 but not when it was 1.0. rubrum ; CTparticles, chromatophore membranes depleted of the soluble transhydrogenase component by repeated washing; dp, proton electrochemical potential gradient; ApH, transmembrane pH gradient; d y , transmembrane electrical potential.