Coupling Hydride Transfer to Proton Pumping: the Swiveling Mechanism of Transhydrogenase

Abstract

The membrane-bound nicotinamide nucleotide transhydrogenase is a key enzyme for the maintenance of metabolic balance in mammalian cells as well as in many bacteria. The enzyme resides in the mitochondrial inner membrane in eukaryotic cells or the cytoplasmic membrane in bacteria. Under normal physiological conditions, the transhydrogenase utilizes the proton motive force to drive hydride transfer from NADH to NADP+, thus generating NADPH. Among other functions, NADPH is critical for the cellular defense against reactive oxygen species. Although not the only source of NADPH, the transhydrogenase is often important, depending on cell type and physiological state. People with the most severe mutations in the Nnt gene, encoding transhydrogenase, suffer from familial glucocorticoid deficiency. Recent X-ray structures of the transhydrogenase from the hyperthermophilic bacterium Thermus thermophilus have provided key insights into how this enzyme couples proton flux across the membrane to hydride transfer. The central hypothesis from these studies focuses on the proposal that large motions of the NADP(H) binding domain (dIII), swiveling between alternating states during the catalytic cycle, are responsible for gating the proton channel in response to the redox state of bound NADP+/NADPH.

Ligand binding and conformational dynamics of the E. coli nicotinamide nucleotide transhydrogenase revealed by hydrogen/deuterium exchange mass spectrometry

“…The energy-coupled transhydrogenase is found in a variety of prokaryotes and in the inner mitochondrial membrane of most eukaryotes, except plants and yeasts [1] , [2] . It plays an important role in cellular processes such as reducing oxidative stress, anabolic reactions and apoptosis [1] , [2] , [3] .…”

“…The energy-coupled transhydrogenase is found in a variety of prokaryotes and in the inner mitochondrial membrane of most eukaryotes, except plants and yeasts [1] , [2] . It plays an important role in cellular processes such as reducing oxidative stress, anabolic reactions and apoptosis [1] , [2] , [3] . As a membrane-embedded protein complex, the transhydrogenase utilizes the proton motive force (PMF) present across the membrane to generate NADPH while transporting one proton from the positive side of the membrane (mitochondrial intermembrane space or bacterial periplasm) to the negative side (mitochondrial matrix or bacterial cytoplasm).…”

“…The membrane-bound transhydrogenase from different species has a conserved structure consisting of two identical protomers in which each protomer has three domains [1] , [3] , schematically illustrated in Fig. 1 A. Domain I contains the NAD(H) binding site whereas NADP(H) binds in domain III [4] , [5] , [6] , [7] .…”

“…1 A. Domain I contains the NAD(H) binding site whereas NADP(H) binds in domain III [4] , [5] , [6] , [7] . These two domains can form an interface where direct hydride transfer from NADH to NADP + (forward reaction) occurs [1] , [6] , [7] , [8] . Domain II is the transmembrane segment which contains the putative proton channel with a single protonatable histidine residue located near the middle of the membrane [1] , [6] .…”

“…These two domains can form an interface where direct hydride transfer from NADH to NADP + (forward reaction) occurs [1] , [6] , [7] , [8] . Domain II is the transmembrane segment which contains the putative proton channel with a single protonatable histidine residue located near the middle of the membrane [1] , [6] . Each protomer has the three domains stacked with domain III (NADP(H)-binding) in between domain II (proton channel) and domain I (NAD(H)-binding).…”