His75-Asp97 Cluster in Green Proteorhodopsin

摘要

The proteorhodopsin (PR) family found in bacteria near the ocean's surface consists of hundreds of PR variants color-tuned to their environment. PR contains a highly conserved single histidine at position 75, which is not found in most other retinal proteins. Using ~(13)C and ~(15)N 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 pK_a. The functional role of His75 has been studied using site-directed mutagenesis and time-resolved optical spectroscopy: Ultrafast vis-pump/vis-probe experiments on PR_(H75N) 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 the photocycle in wild-type PR This means that PR was not optimized by evolution for fast proton transfer, which raises questions about its true function in vivo.