Fourier transform infrared evidence for early deprotonation of Asp⁸⁵ at alkaline pH in the photocycle of bacteriorhodopsin mutants containing E194Q

The role of the extracellular Glu side chains of bacteriorhodopsin in the proton transport mechanism has been studied using the single mutants E9Q, E74Q, E194Q, and E204Q; the triple mutant E9Q/E194Q/E204Q; and the quadruple mutant E9Q/E74Q/E194Q/E204Q. Steady-state difference and deconvoluted Fourier transform infrared spectroscopy has been applied to analyze the M- and N-like intermediates in membrane films maintained at a controlled humidity, at 243 and 277 K at alkaline pH. The mutants E9Q and E74Q gave spectra similar to that of wild type, whereas E194Q, E9Q/E194Q/E204Q, and E9Q/E74Q/E194Q/E204Q showed at 277 K a N-like intermediate with a single negative peak at 1742 cm-1, indicating that Asp85 and Asp96 are deprotonated. Under the same conditions E204Q showed a positive peak at 1762 cm-1 and a negative peak at 1742 cm-1, revealing the presence of protonated Asp85 (in an M intermediate environment) and deprotonated Asp96. These results indicate that in E194Q-containing mutants, the second increase in the Asp85 pK(a) is inhibited because of lack of deprotonation of the proton release group. Our data suggest that Glu194 is the group that controls the pK(a) of Asp85.