Krokinobacter eikastus rhodopsin 2 (KR2) is a recently identified light-driven Na(+) pump from a marine bacterium. KR2 pumps Na(+) in NaCl solution but pumps H(+) in the absence of Na(+) and Li(+). The Na(+) transport mechanism in KR2 has been extensively studied, whereas understanding of the H(+) transport mechanism is very limited. Here we studied ion uptake mechanisms and H(+)-Na(+) selectivity using flash photolysis. The results show that decay of the blue-shifted M intermediate is dependent on both [Na(+)] and [H(+)], indicating that KR2 competitively uptakes Na(+) or H(+) upon M decay. Comprehensive concentration dependence of Na(+) and H(+) revealed that the rate constant of H(+) uptake (kH) was much larger than that of Na(+) uptake (kNa) with a ratio (kH/kNa) of >10(3). Therefore, KR2 pumps only H(+) when Na(+) and H(+) concentrations are similar. On the contrary, KR2 pumps Na(+) exclusively under physiological conditions in which [Na(+)] is much greater than [H(+)].
Keywords: Schiff base; flash photolysis; ion uptake; optogenetics; proton; pump; rate constant; selectivity; sodium ion.