Open AccessStopped‐flow kinetics of hydride transfer between nucleotides by recombinant domains of proton‐translocating transhydrogenase
Author(s) -
Venning Jamie D.,
Bizouarn Tania,
Cotton Nick P. J.,
Quirk Philip G.,
Jackson J. Baz
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2570202.x
Subject(s) - rhodospirillum rubrum , chemistry , nad+ kinase , membrane , hydride , conformational change , dehydrogenase , dissociation (chemistry) , nucleotide , stereochemistry , biophysics , biochemistry , crystallography , enzyme , biology , metal , organic chemistry , gene
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 + /NADH, and domain III for NADP + /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 + analogue, acetylpyridine adenine dinucleotide (AcPdAD + ). 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 + ( k ≈0.03 s −1 ). Phase A of the burst ( k ≈600 s −1 ) probably arises from fast hydride transfer in complexes of domains I and III. Phase B ( k ≈10−50 s −1 ), 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 release. A comparison of the temperature dependences of AcPdAD + reduction by [4B‐ 2 H]NADPH, and by [4B‐ 1 H]NADPH, during phase A shows that there may be a contribution from quantum mechanical tunnelling to the process of hydride transfer. Given that hydride transfer between the nucleotides is direct [Venning, J. D., Grimley, R. L., Bizouarn, T., Cotton, N. P. J. & Jackson, J. B. (1997) J. Biol. Chem. 272 , 27 535−27 538], this suggests very close proximity of the nicotinamide rings of the two nucleotides in the I :III complex.