Transhydrogenase comprises three domains. Domains I and I11 are peripheral to the membrane and possess the NAD(H)-and NADP(H)-binding sites, respectively, and domain 11 spans the membrane. Domain TIT of transhydrogenase from Rhodospirillum rubrum was expressed at high levels in Escherichia coli, and purified. The purified protein was associated with substoichiometric quantities of tightly bound NADP' and NADPH. Fluorescence spectra of the domain 111 protein revealed emissions due to Tyr residues. Energy transfer was detected between Tyr residue(s) and the bound NADPH, indicating that the amino acid residue(s) and the nucleotide are spatially close. The rate constants for NADP' release and NADPH release from domain Ill were 0.03 SKI and S.6X10-4s-1, respectively. In the absence of domain I1 a mixture of the recombinant domain 111 protein, plus the previously described recombinant domain 1 protein, catalysed reduction of acetylpyridine-adenine dinucleotide (AcPdAD') by NADPH (reverse transhydrogenation) at a rate that was limited by the release of NADP' from domain 111. Similarly, the mixture catalysed reduction of thio-NADP' by NADH (forward transhydrogenation) at a rate limited by release of thio-NADPH from domain 111. The mixture also catalysed very rapid reduction of AcPdAD' by NADH, probably by way of a cyclic reaction mediated by the tightly bound NADP(H). Measurement of the rates of the transhydrogenation reactions during titrations of domain I with domain 111 and vice versa indicated (a) that during reduction of AcPdAD' by NADPH, a single domain I protein can visit and transfer H ~ equivalents to about 60 domain 111 proteins during the time taken for a single domain I11 to release its NADP ' , whereas (b) the cyclic reaction is rapid on the timescale of formation and breakdown of the domain I . 111 complex. The rate of the hydride transfer reaction was similar in thc domain I I I11 complex to that in the complete membrane-bound transhydrogenase, but the rates of forward and reverse transhydrogenation were much slower in the I . 111 complex due to the greatly decreased rates of release of NADP' and NADPH. It is concluded that, in the complete enzyme, conforniational changes in the membrane-spanning domain 11, which result from proton translocation, lead to changes in the binding affinity of domain 111 for NADP' and for NADPH.

The redox step in the transhydrogenase reaction is readily visualized; the NC4 atoms of the nicotinamide rings of the bound nucleotides are brought together to facilitate direct hydride transfer with A-B stereochemistry. The asymmetry of the dI:dIII complex suggests that in the intact enzyme there is an alternation of conformation at the catalytic sites associated with changes in nucleotide binding during proton translocation.

H'-transhydrogenase (H'-Thase) and NADP-linked isocitrate dehydrogenase (NADP-ICDH) are very active in animal mitochondria but their physiological function is only poorly understood. This is especially so in the case of the heart and muscle, where there are no major consumers of NADPH. We propose here that H'-Thase and NADP-ICDH have a combined function in the fine regulation of the activity of the tricarboxylic acid (TCA) cycle, providing enhanced sensitivy to changes in energy demand. This is achieved through cycling of substrates by NAD-linked ICDH, NADP-linked ICDH and H'-Thase. It is proposed that NAD-ICDH operates in the forward direction of the TCA cycle, but NADP-ICDH is driven in reverse by elevated levels of NADPH resulting from the action of the transmembrane proton electrochemical potential gradient (dp) on H'-Thase. This has the effect of increasing the sensitivity to allosteric modifiers of NAD-ICDH (NADH, ADP, ATP, Ca" etc), potentially giving rise to large changes in the net flux from iso-citrate to a-ketoglutarate. Furthermore, changes in the level of Ap resulting from changes in the demand for ATP would, via H'-Thase, shift the redox state of the NADP pool and this, in turn, would lead to a change in the rate of the reaction catalysed by NADP-ICDH and hence to an additional and complementary effect on the net metabolic flux from isocitrate to a-ketoglutarate.Other consequences of this substrate cycle are, (i) the production of heat at the expense of Ap, which may contribute to thermoregulation in the animal, and (ii) an increased rate of dissipation of Ap (leak).

Research shows a strong link between adult attachment and mental and physical health, but little is known about the mechanisms that underlie these relationships. The present study examined self-compassion and mattering, two constructs from positive psychology literature, as potential mediators. Using survey data from a sample of 208 college students, relationships among attachment, self-compassion, mattering, and functional health were explored. Correlational analyses indicated that attachment anxiety and avoidance were strongly related to the mental health component of functional health. Mediation analyses indicated that mattering and self-compassion mediated the relationships between attachment orientation (i.e., levels of avoidance and anxiety) and mental health. These findings suggest that individuals' abilities to be kind toward themselves and their sense of belonging and being important to others are pathways through which attachment orientation relates to mental health.

Helix D/loop D interacts with the bound nucleotide and loop E, and probably interacts with the membrane-spanning dII. Changes in ionisation and conformation in helix D/loop D, resulting from proton translocation through dII, are thought to be responsible for the changes in affinity of dIII for NADP(+) and NADPH that drive the reaction.