The His-75-Asp-97 Cluster in Proteorhodopsin: A DNP and Solid-State NMR Study

The proteorhodopsin (PR) family found in bacteria near the ocean's surface to high abundance consists of hundreds of PR molecules which are colour-tuned to their environment. The green absorbing species has been shown to act as a light-driven proton pump in vitro and in non native host organisms. In contrast to bacteriorhodopsin, the pKa of the primary proton acceptor Asp-97 is highly elevated and a single His is found close to the active site. Here, we report a complete biophysical study of this His-75, which is highly conserved within the PR family but not found in other retinal proteins. 13C-13C dipolar correlation experiments did show, that both side chains are close enough to enable the formation of a H-bond. In addition, 15N-MAS NMR provides evidence, that indeed a neutral, H-bonded His-Asp complex is formed in which the protonation is shifted to D97 explaining its high pKa. Using the sensitivity enhancement provided by dynamic nuclear polarisation, we were able characterise the active site and especially the H-bonding character of the His-Asp coupling in great detail. Our study is complemented by site-directed mutagenesis in combination with black lipid membrane measurements, ultrafast optical spectroscopy and flash photolysis. Our data show, that His-75 forms a pH dependent complex with Asp-97 but replacing His-75 with Met, Trp or Asn accelerates PR's photocycle and does not prevent proton transfer. This raises the question of the true function of PR in vivo.