Abstract: Site-directed mutation technique was used to mutate L93/L94 (leucine 93/leucine 94) in bR/aR4 (bacteriorhodopsin/archearhodopsin-4) to alanine and phenylalanine with different side chains, and threonine with different polarities, respectively. The corresponding bR, BI-aR4 (bacterioruberin-included), and BE-aR4 (bacterioruberin-excluded) mutants were obtained. UV-visible absorption spectroscopy, flash photolysis spectroscopy, and pH titration measurements were used to study the effects of L94 on the retinal, photocycle, proton pumping, and the relation between L94 and bacterioruberin. The results showed that L94 regulated the conformation and dynamic equilibrium of the retinal and the helical distribution of the proton channel through the steric hindrance in aR4 to maintain the retinal cis-trans isomerization and re-isomerization and promote proton release. In addition, L94 seemed to be modulated by bacterioruberin. The L94 mutants perturb helixes more strongly in BE-aR4, resulting in changing the hydrogen bond network of the proton donor and proton release cluster.

Key words: archearhodopsin-4, light-driven proton pump, leucine 94, bacterioruberin, bacteriorhodopsin