STRUCTURAL AND CATALYTIC PROPERTIES OF THE EXPRESSED AND PURIFIED NAD(H)-BINDING AND NADP(H)-BINDING DOMAINS OF PROTON-PUMPING TRANSHYDROGENASE FROM ESCHERICHIA-COLI

Proton-pumping nicotinamide nucleotide transhydrogenase from Escherich ia coli contains three domains: the hydrophilic domains I and III harb or the binding sites for NAD(H) and NADP(H), respectively, and domain II represents the membrane-spanning region. Proton translocation invol ves primarily domain II but possibly also domain III, which contains t he essential beta Asp392 residue. In the present investigation, the ma jor portions of domain I (EcTHS alpha 1 and EcTHS alpha 2) and domain III (EcTHS beta 1) were overexpressed in E. coli and purified therefro m. EcTHS beta 1 was purified mainly in its holoform containing approxi mately 95% NADP(+) and 5% NADPH. When combined, EcTHS alpha 1/EcTHS al pha 2 and EcTHS beta 1 were catalytically active, indicating native-li ke structures. Due to the lack of structural information and its possi ble role in proton pumping, EcTHS beta 1 was primarily characterized. Substrate-binding characteristics and conformational changes were inve stigated by fluorescence and CD. Fluorescence arising from the single beta Trp415 of EcTHS beta 1 was quenched upon binding of NADPH by reso nance energy transfer, an effect that provides an important tool for i nvestigating substrate interactions with this domain and the determina tion of K-d values. The apparent relative binding affinity for NADPH w as found to be about 50 times higher than that for NADP(+). Circular d ichroism was used to estimate secondary structure content and for conf ormational analysis of EcTHS beta 1 in the absence and presence of add ed substrates at various temperatures. Results show that domain III co mplexed with NADPH or NADP(+) adopts different conformations. Isoelect ric focusing and native gel electrophoresis experiments support this f inding. It is proposed that these structural differences play a centra l role in a conformationally-driven proton pump mechanism of the intac t enzyme.