Photocycle-dependent conformational changes in the proteorhodopsin cross-protomer Asp-His-Trp triad revealed by DNP-enhanced MAS-NMR.

Proteorhodopsin (PR) is a highly abundant, pentameric, light-driven proton pump. Proton transfer is linked to a canonical photocycle typical for microbial ion pumps. Although the PR monomer is able to undergo a full photocycle, the question arises whether the pentameric complex formed in the membrane via specific cross-protomer interactions plays a role in its functional mechanism. Here, we use dynamic nuclear polarization (DNP)-enhanced solid-state magic-angle spinning (MAS) NMR in combination with light-induced cryotrapping of photointermediates to address this topic. The highly conserved residue H75 is located at the protomer interface. We show that it switches from the (τ)- to the (π)-tautomer and changes its ring orientation in the M state. It couples to W34 across the oligomerization interface based on specific His/Trp ring orientations while stabilizing the pKa of the primary proton acceptor D97 within the same protomer. We further show that specific W34 mutations have a drastic effect on D97 and proton transfer mediated through H75. The residue H75 defines a cross-protomer Asp-His-Trp triad, which potentially serves as a pH-dependent regulator for proton transfer. Our data represent light-dependent, functionally relevant cross talk between protomers of a microbial rhodopsin homo-oligomer.