Biophysical Society Conference | Tahoe 2023

Proton Reactions: From Basic Science to Biomedical Applications

Sunday Speaker Abstracts

STRUCTURE AND PROPOSED MECHANISM OF THE NICOTINAMIDE NUCLEOTIDE TRANSHYDROGENASE FROM E. COLI Robert B. Gennis 3 ; Jiao Li 1,2 ; Jonathan Zoller 4 ; Lici Schurig-Briccio 3 ; Sangjin Hong 3 ; Martin Eisinger 5 ; Malcolm Anderson 6 ; Melanie Radloff 5 ; kristina desch 4 ; Kai Zhang 2 ; Julian Langer 4 ; Jiapeng Zhu 1 ; 1 Nanjing University, School of Medicine and Integrative Holistic Medicine, Nanjing, China 2 Yale University, Department of Molecular Biophysics and Biochemistry, New Haven, CT, USA 3 University of Illinois at Urbana-Champaign, Biochemistry, Urbana, IL, USA 4 Max Planck Institute of Biophysics, Proteomics, Frankfurt am Main, Germany 5 Max Planck Institute of Biophysics, Molecular Membrane Biology, Frankfurt am Main, Germany 6 Waters Corporation, Winslow, United Kingdom The nicotinamide nucleotide transhydrogenase is present in the mitochondrial inner membrane and in the cytoplasmic membranes of many bacteria. The enzyme couples the proton motive force across the membrane to the hydride transfer between NAD(H) and NADP(H). Under most circumstances the enzyme generates NADPH and for each NADPH formed, one proton is translocated across the membrane from the outside (electrically positive) to the inside (electrically negative). The enzyme consists of three domains: domain I binds NAD(H); domain III binds NADP(H); and domain II which contains multiple transmembrane helices and a proton channel. In this work, the structure of the transhydrogenase from E. coli has been determined by cryo-electron microscopy, revealing major conformational changes depending on the presence/absence of nucleotides. These conformational changes were further characterized using hydrogen-deuterium exchange with mass spectrometry (HDX-MS). Results show coupling of the conformation of the helices comprising the transmembrane proton channel in domain II to the binding of either NADPH or NADP+ to domain III. The data are generally consistent with the mechanism previously proposed by Kampjut and Sazanov [Nature (2019) 573, 291] based on studies of the ovine transhydrogenase. The proton channel contains a single protonatable histidine which can be exposed either to the periplasmic side or the cytoplasmic side of the membrane. The side to which the histidine is exposed as well as its pK a is determined by whether the reactant (NADP+) or product (NADPH) is bound to domain III. The transhydrogenase functions essentially as a ligand-gated proton transporter.

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