Efficient nanomaterials for artificial photosynthesis require fast and robust unidirectional electron transfer (ET) from photosensitizers through charge-separation and accumulation units to redox-active catalytic sites. We explored the ultrafast time-scale limits of photo-induced charge transfer between a Ru(II)tris(bipyridine) derivative photosensitizer and PpcA, a 3-heme c-type cytochrome serving as a nanoscale biological wire. Four covalent attachment sites (K28C, K29C, K52C, and G53C) were engineered in PpcA enabling site-specific covalent labeling with expected donor-acceptor (DA) distances of 4–8 Å. X-ray scattering results demonstrated that mutations and chemical labeling did not disrupt the structure of the proteins. Time-resolved spectroscopy revealed three orders of magnitude difference in charge transfer rates for the systems with otherwise similar DA distances and the same number of covalent bonds separating donors and acceptors. All-atom molecular dynamics simulations provided additional insight into the structure-function requirements for ultrafast charge transfer and the requirement of van der Waals contact between aromatic atoms of photosensitizers and hemes in order to observe sub-nanosecond ET. This work demonstrates opportunities to utilize multi-heme c-cytochromes as frameworks for designing ultrafast light-driven ET into charge-accumulating biohybrid model systems, and ultimately for mimicking the photosynthetic paradigm of efficiently coupling ultrafast, light-driven electron transfer chemistry to multi-step catalysis within small, experimentally versatile photosynthetic biohybrid assemblies.

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Molecular docking is a widely used computational method to mimic ligandprotein association in silico. However, predictions are largely affected by the type and extent of protein conformational changes accompanying ligand binding. Among the various receipts developed to address the ''flexibility issue,'' one of the more popular is ensemble-docking. In ensemble-docking, a set of different conformations of the protein, obtained either experimentally or from computer simulations, are used altogether in order to implicitly account for structural deformations induced by ligand binding. While molecular dynamics simulations are largely employed to obtain receptor conformations, sampling hololike structures (that are prone to host ligands) remains challenging, ultimately affecting docking accuracy. In order to address this limitation, we introduce EDES -Ensemble-Docking with Enhanced-sampling of pocket Shape -, a computational approach based on metadynamics simulations which successfully generates holo-like conformations of proteins only exploiting their apo structures. This is achieved by defining a set of collective variables that effectively sample different shapes of the binding site, ultimately mimicking the steric effect due to ligands. We assessed the method on several challenging proteins undergoing different extents of conformational changes upon ligand binding (from 1 A ˚for recombinant ricin to 7 A ˚for adenylate kinase), as well as on the Grand Challenge 4 blind prediction competition. In all cases, our protocol generated a significant fraction of structures featuring a low RMSD from the experimental holo geometry. Moreover, ensemble docking calculations using those conformations yielded in all cases native-like poses among the top-ranked ones.

Light-driven water oxidation in algae, cyanobacteria and higher plants generates dioxygen that supports life on Earth. The water-oxidation reaction is catalyzed by the oxygen-evolving complex (OEC) in photosystem II (PSII) that is comprised of the tetranuclear manganese calcium-oxo (Mn 4 Ca-oxo) cluster, redox-active tyrosine residue (Y Z ) and hydrogen-bonded network of amino acids and water molecules. The redox-active tyrosine residue, Y Z , mediates successive proton-coupled electron transfer (PCET) reactions that are essential for the oxidation of water to dioxygen at the Mn 4 Ca-oxo cluster. It is proposed that the strong hydrogen bond between Y Z and and its conjugate base, D1-His190, likely renders Y Z kinetically competent, leading to highly efficient water oxidation. However, a detailed understanding of PCET at Y Z remains elusive due to the lack of high-resolution structural methods to directly probe the electron-and proton-transfer reactions. In this study, we utilize highresolution two-dimensional (2D) 14 N and 1 H hyperfine sublevel correlation (HYSCORE) spectroscopy to investigate the electronic structure of the bioinspired artificial reaction center, benzimidazole-phenol porphyrin, that mimics PCET at the Y Z residue of PSII. Additionally, we perform density functional theory (DFT) calculations to determine the electron spin density distribution and electron-nuclear hyperfine coupling parameters of the benzimidazolephenol porphyrin radical for comparison with the corresponding parameters that are obtained from the 2D HYSCORE measurements. Redox-active proteins covalently modified with photosensitizers have served as important model systems for elucidation of kinetic principles controlling biological electron transfer. To date, the fastest rates observed in these systems were 10ns and involved donor acceptor (DA) distances of >0.8nm. This leaves a significant gap in rates and distances for charge transfer rates occurring in Nature but poorly accessible in experimental studies. To this end, we developed a series of 22 cysteine mutants of PpcA, a 3-heme cytochrome from Geobacter sulfurreducens. Due to its relatively small size (71 amino acids) we expected to obtain a number of mutants with DA distance of 0.4-0.5nm. These proteins were successfully expressed in E.coli and isolated for covalent labeling with Ru(bpy)2(bpy-Br). With time-resolved nanosecond and ultrafast transient absorbance spectroscopy we have identified 6 constructs with apparent photo-induced charge transfer time constants of 20 ps or faster, including 2 constructs with 1-2 ps time constants. The is a significant result as up to this point only natural photosynthetic systems demonstrated such a fast initial charge separation, while all artificial covalent biohybrid constructs exhibited charge transfer rates 3 or more orders of magnitude slower. To understand molecular principles responsible for such a dramatic acceleration of electron transfer rates, we used small-and wide angle X-ray scattering and currently attempting to obtain X-ra...