Titration of E. coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range

A pH-titration 2D NMR study of Escherichia coli transhydrogenase domain III with bound NADP+ or NADPH has been carried out, in which the pH was varied between 5.4 and 12. In this analysis, individual amide protons served as reporter groups. The apparent pKa values of the amide protons, determined from the pH-dependent chemical shift changes, were attributed to actual pKa values for several titrating residues in the protein. The essential Asp392 is shown to be protonated at neutral pH in both the NADP+ and NADPH forms of domain III, but with a marked difference in pKa not only attributable to the charge difference between the substrates. Titrating residues found in loop D/α5 point to a conformational difference of these structural elements that is redox-dependent, but not pH dependent. The observed apparent pKa values of these residues are discussed in relation to the crystal structure of Rhodospirillum rubrum domain III, the solution structure of E. coli domain III and the mechanism of intact proton-translocating transhydrogenase.