Structural and catalytic properties of the expressed and purified NAD(H)- and NADP(H)-binding domains of proton-pumping transhydrogenase from Escherichia coli

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains three domains: the hydrophilic domains I and III harbor the binding sites for NAD(H) and NADP(H), respectively, and domain II represents the membrane-spanning region. Proton translocation involves primarily domain II but possibly also domain III, which contains the essential beta Asp392 residue. In the present investigation, the major portions of domain I (EcTHS alpha 1 and EcTHS alpha 2) and domain III (EcTHS beta 1) were overexpressed in E. coli and purified therefrom. EcTHS beta 1 was purified mainly in its holoform containing approximately 95% NADP(+) and 5% NADPH. When combined, EcTHS alpha 1/EcTHS alpha 2 and EcTHS beta 1 were catalytically active, indicating native-like structures. Due to the lack of structural information and its possible role in proton pumping, EcTHS beta 1 was primarily characterized. Substrate-binding characteristics and conformational changes were investigated by fluorescence and CD. Fluorescence arising from the single beta Trp415 of EcTHS beta 1 was quenched upon binding of NADPH by resonance energy transfer, an effect that provides an important tool for investigating substrate interactions with this domain and the determination of K-d values. The apparent relative binding affinity for NADPH was found to be about 50 times higher than that for NADP(+). Circular dichroism was used to estimate secondary structure content and for conformational analysis of EcTHS beta 1 in the absence and presence of added substrates at various temperatures. Results show that domain III complexed with NADPH or NADP(+) adopts different conformations. Isoelectric focusing and native gel electrophoresis experiments support this finding. It is proposed that these structural differences play a central role in a conformationally-driven proton pump mechanism of the intact enzyme.