Proton diffusion along biological membranes is vitally important for cellular energetics. Here we extended previous time-resolved fluorescence measurements to study the timeandtemperature dependence of surface proton transport. We determined the Gibbs activation energy barrier ΔG^‡_rthat opposes proton surface-to-bulk release from Arrhenius plots of (i) protons’ surface diffusion constant and (ii) the rate coefficient for proton surface-to-bulk release. The large size of ΔG^‡_rdisproves that quasi-equilibrium exists in our experiments between protons in the near-membrane layers and in the aqueous bulk. Instead, non-equilibrium kinetics describes the proton travel between the site of its photo-release and its arrival at a distant membrane patch at different temperatures. ΔG^‡_rcontains only a minor enthalpic contribution that roughly corresponds to the breakage of a single hydrogen bond. Thus, our experiments reveal an entropic trap that ensures channeling of highly mobile protons along the membrane interface in the absence of potent acceptors.

A 2,2′-bipyridyl-containing poly(arylene-ethynylene)-alt-poly(arylene-vinylene) polymer, acting as a light-harvesting ligand system, was synthesized and coupled to an organometallic rhodium complex designed for photocatalytic NAD⁺/NADH reduction. The material, which absorbs over a wide spectral range, was characterized by using various analytical techniques, confirming its chemical structure and properties. The dielectric function of the material was determined from spectroscopic ellipsometry measurements. Photocatalytic reduction of nucleotide redox cofactors under visible light irradiation (390–650 nm) was performed and is discussed in detail. The new metal-containing polymer can be used to cover large surface areas (e.g. glass beads) and, due to this immobilization step, can be easily separated from the reaction solution after photolysis. Because of its high stability, the polymer-based catalyst system can be repeatedly used under different reaction conditions for (photo)chemical reduction of NAD⁺. With this concept, enzymatic, photo-biocatalytic systems for solar energy conversion can be facilitated, and the precious metal catalyst can be recycled.

Hydrogenases are metalloenzymes that catalyze the conversion of protons and molecular hydrogen, H2. [FeFe]-hydrogenases show particularly high rates of hydrogen turnover and have inspired numerous compounds for biomimetic H2 production. Two decades of research on the active site cofactor of [FeFe]-hydrogenases have put forward multiple models of the catalytic proceedings. In comparison, our understanding of the catalytic proton transfer is poor. Previously, residues were identified forming a proton transfer pathway between active site cofactor and bulk solvent; however, the exact mechanism of catalytic proton transfer remained inconclusive. Here, we employ in situ infrared difference spectroscopy on the [FeFe]-hydrogenase from Chlamydomonas reinhardtii evaluating dynamic changes in the hydrogen-bonding network upon photoreduction. While proton transfer appears to be impaired in the oxidized state (Hox), the presented data support continuous proton transfer in the reduced state (Hred). Our analysis allows for a direct, molecular unique assignment to individual amino acid residues. We found that transient protonation changes of glutamic acid residue E141 and, most notably, arginine R148 facilitate bidirectional proton transfer in [FeFe]-hydrogenases.