Roles of cytoplasmic arginine and threonine in chloride transport by the bacteriorhodopsin mutant D85T

In the light-driven anion pump halorhodopsin (HR), the residues arginine 20 0 and threonine 203 are involved in anion release at the cytoplasmic side o f the membrane. Because of large sequence homology and great structural sim ilarities between HR and bacteriorhodopsin (BR), it has been suggested that anion translocation by HR and by the chloride-pumping BR mutant BR-D85T oc curs by the same mechanism. Consequently, the functions of the R200/T203 pa ir in HR should be the same as those of the corresponding pair in BR-D85T ( R175/T178). We have put this hypothesis to a test by creating two mutants o f BR-D85T in which R175 and T178 were replaced by glutamine and valine, res pectively. Chloride transport activities were essentially the same for all three mutants. whereas chloride binding and the kinetics of parts of the ph otocycle were markedly affected by the replacement of T178. In contrast, th e consequences of mutating R175 proved to be less significant. These findin gs are consistent with evidence obtained on HR and therefore support the id ea that the respective mechanistic roles of the cytoplasmic arginine/threon ine pairs in HR and BR-D85T are equal.