Buffer Effects on Electric Signals of Light-Excited Bacteriorhodopsin

Abstract

Buffers change the electric signals of light-excited bacteriorhodopsin molecules in purple membrane if their concentration and the pH of the low-salt solution are properly selected. "Positive" buffers produce a positive component, and "negative" buffers a negative component in addition to the signals due to proton pumping. Measurement of the buffer effects in the presence of glycyl-glycine or bis-tris propane revealed an increase of approximately 2 and a change of sign and a decrease to approximately -0.5 in the translocated charge in these cases, respectively. These factors do not depend on temperature. The Arrhenius parameters established from the evaluation of the kinetics indicate activation enthalpies of 35-40 kJ/mol and negative activation entropies for the additional signals. These values agree with those found by surface-bound pH-sensitive probes in the search of the timing of proton release and uptake. The electric signals were also measured in the case of D(2)O solutions with similar results, except for the increased lifetimes. We offer a unified explanation for the data obtained with surface-bound probes and electric signals based on the clusters at extracellular and cytoplasmic sites of bacteriorhodopsin participating in proton release and uptake.

Excitation of the M Intermediates of Bacteriorhodopsin†

“…It has been demonstrated earlier that the opposite process ( i.e. proton release to the bulk in the normal photocycle) is accompanied by a conformational change in the PRG (42). Based on this, and the similar kinetics of the signals in Fig.…”

“…We found earlier that PERS of light‐excited BR measured with and without GG buffer differ in the microsecond time range (42–44). Shortly, the difference between these signals showed that they originated from the protons that were released at the extracellular surface by the PRG and moved on the buffer gradient (42). This “buffer signal” reflects the appearance of protons in solution, consequently we consider it as a differential response of proton release ( i.e.…”

Actinic Light-Energy Dependence of Proton Release from Bacteriorhodopsin

“…On the contrary, the ''buffer effect'' responds to the protons as released. The protein electric response signals (PERS) of light excited BR measured with and without GG differ in the microsecond time range (6,7,16,17). Detailed study of the difference between these signals showed that they originated from the protons that were released at the extracellular surface by PRG and moved on the buffer gradient (6).…”

“…The buffer signal has rising and decaying components with lifetimes of ;50 and 250 ms. The interpretation of these two components is given in Tóth-Boconádi et al (6). Shortly, protons move from the Schiff-base to Asp-85 during L/M transition; the change of electric field rearranges the proton release group (rise of buffer signal), which then releases the proton (decay of buffer signal).…”

“…We set out to study the fluence dependence of proton release from pm. Two methods were applied: the usual proton indicator dye pyranine and the ''buffer method'' developed in our laboratory (6,7). We found by both methods that the lifetimes of proton release and uptake increase with increasing fluence, whereas the proton pumping activity decreases.…”