Evidence for intraprotein charge transfer during the transport activity ofthe melibiose permease from Escherichia coli

Electrogenic activity associated with the activity of the melibiose permeas e (MelB) of Escherichia coli was investigated by using proteoliposomes cont aining purified MelB adsorbed onto a solid-supported membrane. Transient cu rrents were selectively recorded by applying concentration jumps of Na+ ion s (or Li+) and/or of different sugar substrates of MelB (melibiose, thio-me thyl galactoside, raffinose) using a fast-flow solution exchange system. Ch aracteristically, the transient current response was fast, including a sing le decay exponential component (tau approximate to 15 ms) on applying a Na (or Li+) concentration jump in the absence of sugar. On imposing a Na+ (or Li+) jump on proteoliposomes preincubated with the sugar, a sugar jump in a preparation preincubated with the cation, or a simultaneous jump of the c ation and sugar substrates, the electrical transients were biphasic and com prised both the fast and an additional slow (tau approximate to 350 ms) dec ay components. Finally, selective inactivation of the cosubstrate transloca tion step by acylation of MelB cysteins with N-ethyl maleimide suppressed t he slow response components and had no effect on the fast transient one. We suggest that the fast transient response reflects charge transfer within M elB during cosubstrate binding while the slow component is associated with charge transfer across the proteoliposome membrane. From the time course of the transient currents, we estimate a rate constant for Na+ binding in the absence and presence of melibiose of k > 50 s(-1) and one for melibiose bi nding in the absence of Na+ of k approximate to 10 s(-1).