Roles of Individual Amino Acids in Helix 14 of the Membrane Domain of Proton-Translocating Transhydrogenase from Escherichia coli As Deduced from Cysteine Mutagenesis

Abstract

Proton-translocating nicotinamide nucleotide transhydrogenase is a membrane-bound protein composed of three domains: the hydrophilic NAD(H)-binding domain, the hydrophilic NADP(H)-binding domain, and the hydrophobic membrane domain. The latter harbors the proton channel. In Escherichia coli transhydrogenase, the membrane domain is composed of 13 transmembrane alpha helices, of which especially helices 13 and 14 contain conserved residues. To characterize the roles of the individual residues betaLeu240 to betaSer260 in helix 14, these were mutated as single mutants to cysteines in the cysteine-free background, and in the case of betaGly245, betaGly249, and betaGly252, also to leucines. In addition to the residues forming the helix, residues betaAsn238 and betaAsp239 were also mutated. Except for betaI242C, all mutants were normally expressed, purified, and characterized with respect to, e.g., catalytic activities and proton pumping. The results show that mutation of the conserved glycines betaGly245, betaGly249, and betaGly252, located on the same face of the helix, led to a general inhibition of all activities, especially in the case of betaGly252, suggesting a role of these glycines in helix-helix interactions. In contrast, mutation of the conserved serines betaSer250, betaSer251, and betaSer256 led to enhanced activities of all reactions, including the cyclic reaction which was mediated by bound NADP(H). Mutation of the remaining residues resulted in intermediate inhibitory effects. The results strongly support an important regulatory role of the membrane domain on the NADP(H)-binding site.

Essential Glycine in the Proton Channel of Escherichia coli Transhydrogenase

“…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.…”

“…In particular, the G245C, G249C, and G252C mutants had significantly reduced reverse transhydrogenation activity (24%, 40%, and Ͻ5%, respectively). Further, the G245L, G249L, and G252L mutants had reverse activities of 22, 70, and 7%, respectively (36). These data are consistent with residues 245, 249, and 252 occurring the same face of helix 14 and indicate that disruption of helical packing within domain II affects proton pumping (36).…”

“…In contrast, both reverse and cyclic activities exceeding that of wild type is observed for the S250A, S251A, S256A, and Y257F mutants (32). 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).…”

“…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.…”