Abstract
We investigated the temperature-dependent kinetics of the light-driven Na(+) pump Krokinobacter rhodopsin 2 (KR2) at Na(+)-pumping conditions. The recorded microsecond flash photolysis data were subjected to detailed global target analysis, employing Eyring constraints and spectral decomposition. The analysis resulted in the kinetic rates, the composition of the different photocycle equilibria, and the spectra of the involved photointermediates. Our results show that with the temperature increase (from 10 to 40°C), the overall photocycle duration is accelerated by a factor of 6, with the L-to-M transition exhibiting an impressive 40-fold increase. It follows from the analysis that in KR2 the chromophore and the protein scaffold are more kinetically decoupled than in other microbial rhodopsins. We link this effect to the rigidity of the retinal protein environment. This kinetic decoupling should be considered in future studies and could potentially be exploited for fine-tuning biotechnological applications.