Thermodynamic Basis of Temperature Adaptation in Three Outward Proton Pump Rhodopsins Distributed Across Diverse Thermal Environments

Microbial rhodopsins with light-driven outward proton pump activity are widely distributed across Earth's diverse environments and contribute to solar energy conversion in global ecosystems. Yet, the thermodynamic principles enabling their function across broad temperature ranges remain poorly understood. Here, we examined the photocycles of three outward proton pump rhodopsins originating from low-, moderate-, and high-temperature environments─HnPR, PR, and TR─and analyzed their kinetics over a wide temperature range using flash photolysis. Thermodynamic activation parameters were determined for each transition state based on transition-state theory. Comparative analysis revealed that the balance of enthalpic and entropic contributions in the early P1 → P2 transition strongly correlates with the environmental temperature in which each rhodopsin is distributed, reflecting distinct adaptation strategies among the three proteins. TR exhibited temperature-dependent alterations in its photocycle, including a shift in the linearity of the Eyring plot for the P2 → P3 transition, consistent with a structural rearrangement previously observed by time-resolved FTIR spectroscopy. In contrast, HnPR displayed hallmark features of cold-adapted proteins in the P4 → P0 transition, including a reduced activation enthalpy and a pronounced decrease in activation entropy, enabling efficient turnover at low temperatures. Together, these findings provide a thermodynamic framework for understanding how outward proton pump rhodopsins function across diverse thermal habitats and illustrate the distinct molecular strategies by which they balance functional dynamics and structural stability to support light-driven proton transport in nature.