Two Consecutive Polar Amino Acids at the End of Helix E are Important for Fast Turnover of the Archaerhodopsin Photocycle.

Archaerhodopsins (ARs) is one of the members of microbial rhodopsins. Threonine 164 (T164) and serine 165 (S165) residues of the AR from Halorubrum sp. ejinoor (HeAR) are fully conserved in ARs, although they are far from the proton transfer channel and the retinal Schiff base, and are likely involved in a hydrogen-bonding network at the end of the Helix E where most microbial rhodopsins assume a "bent structure". In the present work, T164 and/or S165 were replaced with an alanine (A), and the photocycles of the mutants were analyzed with flash photolysis. The amino acid replacements caused profound changes to the photocycle of HeAR including prolonged photocycle, accelerated decay of M intermediate and appearance of additional two intermediates which were evident in T164A- and T164A/S165A-HeAR photocyles. These results suggest that although T164 and S165 are located at the far end of the photoactive center, these two amino acid residues are important for maintaining the fast turnover of the HeAR photocycle. The underlying molecular mechanisms are discussed in relation to hydrogen-bonding networks involving these two amino acids. Present study may arouse our interests to explore the functional role of the well-conserved "bent structure" in different types of microbial rhodopsin.