Identification of a Region Involved in the Communication between the NADP(H) Binding Domain and the Membrane Domain in Proton Pumping E. coli Transhydrogenase

Abstract

The two hydrophilic domains I and III of Escherichia coli transhydrogenase containing the binding sites for NAD(H) and NADP(H), respectively, are located on the cytosolic side of the membrane, whereas the hydrophobic domain II is composed of 13 transmembrane alpha-helices, and is responsible for proton transport. In the present investigation the segment betaC260-betaS266 connecting domain II and III was characterized primarily because of its assumed role in the bioenergetic coupling of the transhydrogenase reaction. Each residue of this segment was replaced by a cysteine in a cysteine-free background, and the mutated proteins analyzed. Except for betaS266C, binding studies of the fluorescent maleimide derivative MIANS to each cysteine in the betaC260-betaR266 region revealed an increased accessibility in the presence of NADP(H) bound to domain III; an opposite effect was observed for betaS266. A betaD213-betaR265 double cysteine mutant was isolated in a predominantly oxidized form, suggesting that the corresponding residues in the wild-type enzyme are closely located and form a salt bridge. The betaS260C, betaK261C, betaA262C, betaM263, and betaN264 mutants showed a pronounced inhibition of proton-coupled reactions. Likewise, several betaR265 mutants and the D213C mutant showed inhibited proton-coupled reactions but also markedly increased values. It is concluded that the mobile hinge region betaC260-betaS266 and the betaD213-betaR265 salt bridge play a crucial role in the communication between the proton translocation/binding events in domain II and binding/release of NADP(H) in domain III.

Essential Glycine in the Proton Channel of Escherichia coli Transhydrogenase

“…This activity depends upon NADPH binding in domain III, whereas in intact TH it is influenced by domain II (30,(32)(33)(34)(35), consistent with a mechanism in which NADPH binding induces conformational change as a basis for enzyme function (Reaction 1) (1-4, 19 -23). With respect to cyclic transhydrogenation, the domain II Gly 3 Ala mutants exhibited 31-96% activity in the presence of added NADP(H) ( Table I).…”

“…Greatly enhanced reverse and cyclic activities were observed for the S250C, S251C, and S256C mutants, consistent with intrinsically bound NADP(H) (36). K m for NADP(H) and the pH dependence of the cyclic reaction were also affected in these mutants (30,33,35,36). These properties may be due to nonnative conformations induced by mutagenesis.…”

“…Because helix 14 is linked at its C terminus to domain III, it may participate in communicating conformational change between domains II and III. Experiments focused on ␤ subunit residues 260 -266 indicate that this occurs with the hinge region (35); recently, helix 14 residues 240 -260 have been mutated to Cys (36). In particular, the G245C, G249C, and G252C mutants had significantly reduced reverse transhydrogenation activity (24%, 40%, and Ͻ5%, respectively).…”

Review and Hypothesis. New insights into the reaction mechanism of transhydrogenase: Swivelling the dIII component may gate the proton channel

“…Conservative single-site substitutions of only EcaH450 (in TM3), EcbH91 (TM9), EcbS139 (TM10), EcbD213 (in the cytoplasmic loop between TM12 and 13), EcbN222 (TM13), EcbG252 (TM14) and in a run of residues from EcbK261-bR265 (in the hinge at the C-terminus of TM14 which connects dII to dIII -see Fig. 1) of many examined in E. coli dII lead to strong inhibition of transhydrogenase activity [30,31,[41][42][43][44][45][46][47][48]. EcbH91 has often provoked interest in that it is the only conserved protonatable residue close to the centre of the membrane dielectric whose substitution significantly deactivates transhydrogenation.…”

“…Low concentrations of Zn ++ block proton translocation through the E. coli dII channel; both FTIR [49] and X-ray absorption studies [50] implicate a His residue in the metal-ion binding but the role of bHis91 in the process is not yet clear. The conserved salt bridge between TtbD202 and TtbR254 in the hinge, which very likely has an important role in catalysis [44], is located next to the proton channel at the membrane surface [22]. Differences in electron density of the hinge region suggest different conformations in the two protomers.…”