Vibrational Study of the Inward Proton Pump Xenorhodopsin NsXeR: Switch Order Determines Vectoriality.

Common proton pumps, e.g. HsBR and PR, transport protons out of the cell. Xenorhodopsins (XeR) were the first discovered microbial rhodopsins which come as natural inward proton pumps. In this work we combine steady-state (cryo-)FTIR and Raman spectroscopy with time-resolved IR and UV/Vis measurements to roadmap the inward proton transport of NsXeR and pinpoint the most important mechanistic features. Through the assignment of characteristic bands of the protein backbone, the retinal chromophore, the retinal Schiff base and D220, we could follow the switching processes for proton accessibility in accordance with the isomerization / switch / transfer model. The corresponding transient IR signatures suggest that the initial assignment of D220 as the proton acceptor needs to be questioned due to the temporal mismatch of the Schiff base and D220 protonation steps. The switching events in the K-L and MCP-MEC transitions are finely tuned by changes of the protein backbone and rearrangements of the Schiff base. This finely tuned mechanism is disrupted at cryogenic temperatures, being reflected in the replacement of the previously reported long-lived intermediate GS* by an actual redshifted (O-like) intermediate.

The Voltage Dependent Sidedness of the Reprotonation of the Retinal Schiff Base Determines the Unique Inward Pumping of Xenorhodopsin.

The new class of microbial rhodopsins, called xenorhodopsins (XeRs),[1] extends the versatility of this family by inward H+ pumps.[2-4] These pumps are an alternative optogenetic tool to the light-gated ion channels (e.g. ChR1,2), because the activation of electrically excitable cells by XeRs is independent from the surrounding physiological conditions. In this work we functionally and spectroscopically characterized XeR from Nanosalina (NsXeR).[1] The photodynamic behavior of NsXeR was investigated on the ps to s time scale elucidating the formation of the J and K and a previously unknown long-lived intermediate. The pH dependent kinetics reveal that alkalization of the surrounding medium accelerates the photocycle and the pump turnover. In patch-clamp experiments the blue-light illumination of NsXeR in the M state shows a potential-dependent vectoriality of the photocurrent transients, suggesting a variable accessibility of reprotonation of the retinal Schiff base. Insights on the kinetically independent switching mechanism could furthermore be obtained by mutational studies on the putative intracellular H+ acceptor D220.

Connectivity matters - ultrafast isomerization dynamics of bisazobenzene photoswitches.

We have investigated the ultrafast dynamics of o-, m- and p-bisazobenzenes, which represent elementary building blocks for photoswitchable multiazobenzene nanostructures. The connectivity pattern within bisazobenzenes and the ensuing complex interactions between the individual azobenzene units determines the ultrafast dynamics of these compounds and their photochemical properties. While retaining a relatively high E → Z isomerization quantum yield, o-bisazobenzene exhibits a very high thermal relaxation rate (half-life of 1.6 ms). Our theoretical calculations reveal that the geometry allows intramolecular excitonic interaction between the azobenzene units, which is reflected in the femtosecond transient absorption data via the simultaneous bleaching of the two excitonic bands. In contrast, the properties of m-bisazobenzene are very similar to the monomeric azobenzene, with the two units acting nearly independently from each other. The highest degree of π conjugation extending over the two azobenzene units was observed for p-bisazobenzene, which results in strong planarity of the molecule, reduced excited state lifetime and relatively low isomerization quantum yield. Multiphotochromic systems bridge the gap between molecular photoswitches and macroscopic function and thus, understanding the properties of bisazobenzenes opens the way to the design and development of new structures with extensive and versatile applications.