Evidence That the Transfer of Hydride Ion Equivalents between Nucleotides by Proton-translocating Transhydrogenase Is Direct

Abstract

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.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 energycoupling 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. 1-3).The results of some early experiments on transhydrogenases from mitochondria and from Rhodospirillum rubrum were interpreted as evidence for the existence of a stable, reducedenzyme intermediate (4 -6). 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,where E(H) represents the reduced-enzyme intermediate. However, in subsequent work other plausible explanations were found for the earlier data (7, 8). Moreover, the conclusions from steady-state kinetic analysis of transhydrogenase from various sources (7, 9 -11) 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 intermediate within the ternary complex, that is a reaction of the following type.The possibility that Cys residues in the polypeptide chain might serve as reducible intermediates in hydride transfer (see, for example, Refs. 1 and 12) 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 (13). 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 ha...

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

“…1 A. The reaction took place in a biphasic burst, before the onset of the steady state, which is limited in rate by very slow release of NADP ϩ [9,12]. In the conditions of the experiment, the steady-state rate of transhydrogenation was equivalent to approximately 0.0004 A · s Ϫ1 , which is negligible on the time scale of the figure.…”

“…The significance of this observation is that it shows that hydride transfer between nucleotides in the recombinant domain I:III complex is fast. It is unlikely, therefore, that the membrane-spanning domain II participates directly in the hydride-transfer process.More recently, it was found by means of stopped-flow spectrophotometry that fast hydride transfer from NADPH to AcPdAD ϩ in the I:III complex could be observed directly as a burst before the reaction becomes limited by NADP ϩ release in the steady state [12]. Two observations led to the conclusion that hydride transfer between the nucleotides is direct, and does not…”

“…More recently, it was found by means of stopped-flow spectrophotometry that fast hydride transfer from NADPH to AcPdAD ϩ in the I:III complex could be observed directly as a burst before the reaction becomes limited by NADP ϩ release in the steady state [12]. Two observations led to the conclusion that hydride transfer between the nucleotides is direct, and does not…”

“…When prepared as described above, the domain III protein was associated with tightly-bound NADP ϩ (typically 0.9 mol · mol Ϫ1 ) and NADPH (about 0.1 mol · mol Ϫ1 ) Ϫ for assay procedures for the tightly-bound nucleotide, see [9]. Before the stopped-flow experiments were performed, bound NADP ϩ was replaced by incubating domain III protein (75 µM) in the above medium supplemented with 105 µM NADPH at 4°C for 2 h [12].…”

“…More recently, it was found by means of stopped-flow spectrophotometry that fast hydride transfer from NADPH to AcPdAD ϩ in the I:III complex could be observed directly as a burst before the reaction becomes limited by NADP ϩ release in the steady state [12]. Two observations led to the conclusion that hydride transfer between the nucleotides is direct, and does not involve any intermediate redox centres: (a) The kinetics of AcPdAD ϩ reduction were similar, on a millisecond time scale, to the kinetics of NADPH oxidation, and (b) analysis by electrospray mass spectrometry showed that there are no covalently bound prosthetic or metal groups associated with either domain I or domain III.…”