Structural and Catalytic Properties of the Expressed and Purified NAD(H)- and NADP(H)-Binding Domains of Proton-Pumping Transhydrogenase from Escherichia coli

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Abstract
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 betaAsp392 residue. In the present investigation, the major portions of domain I (EcTHSalpha1 and EcTHSalpha2) and domain III (EcTHSbeta1) were overexpressed in E. coli and purified therefrom. EcTHSbeta1 was purified mainly in its holoform containing approximately 95% NADP+ and 5% NADPH. When combined, EcTHSalpha1/EcTHSalpha2 and EcTHSbeta1 were catalytically active, indicating native-like structures. Due to the lack of structural information and its possible role in proton pumping, EcTHSbeta1 was primarily characterized. Substrate-binding characteristics and conformational changes were investigated by fluorescence and CD. Fluorescence arising from the single betaTrp415 of EcTHSbeta1 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 Kd 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 EcTHSbeta1 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.

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