The proteorhodopsin (PR) family found in bacteria near the ocean’s surface consists of hundreds of PR variants colour-tuned to their environment. PR contains a highly conserved single histidine at position 75, which is not found in most other retinal proteins. Using 13C- and 15N-MAS-NMR, we were able to prove for green PR that His75 forms a pH dependent H-bond with the primary proton acceptor Asp97, which explains its unusually high pKa. The functional role of His75 has been studied using site directed mutagenesis and time-resolved optical spectroscopy: Ultrafast vis-pump/vis-probe experiments on PRH75N showed that the primary reaction dynamics is retained, while flash photolysis experiments revealed an accelerated photocycle. Our data show the formation of a pH-dependent His-Asp cluster which might be typical for eubacterial retinal proteins. Despite its stabilizing function, His75 was found to slow down the photocycle in wildtype PR. This means that PR was not optimized by evolution for fast proton transfer, which raises questions about its true function in vivo.
- Hempelmann F, Hölper S, Verhoefen MK, Woerner AC, Köhler T, Fiedler SA, Pfleger N, Wachtveitl J, Glaubitz C. His75-Asp97 cluster in green proteorhodopsin. J Am Chem Soc.; 133:4645 (2011).