Artificial Carbonic Anhydrase-Ruthenium Enzyme for Photocatalytic Water Oxidation

Bovine carbonic anhydrase (BCA) is an enzyme that regulates cellular pH by catalyzing CO2 hydration. In this work, we used its well-defined zinc-containing active site to host a series of four sulfonamide-functionalized ruthenium-based water oxidation catalysts Ru1 to Ru4, thereby producing four BCA-Ru1 to BCA-Ru4 artificial metalloenzymes (ArMs). The four ruthenium complexes differed either by the nature of the spectator ligand (bda2– or tda2–) bound to the catalytic center or by the length of the linker between the axially ruthenium-bound pyridine moiety and the zinc-binding sulfonamide. The two ArMs BCA-Ru1 and BCA-Ru2 were catalytically active for photocatalytic water oxidation in aqueous solution in the presence of [Ru(bpy)3](ClO4)2 as a photosensitizer, Na2S2O8 as an electron acceptor, and blue light (450 nm). The most active artificial metalloenzyme, BCA-Ru1, could drive photocatalytic O2 production at particularly low ArM concentrations (5 μM), yielding a turnover number (TON) of 348 and a turnover frequency (TOF) of 9 min–1 that was 1 order of magnitude higher than for the enzyme-free catalyst. A molecular dynamics study was performed to model the interaction between the ruthenium catalyst and the BCA protein. Overall, the protein scaffold modified the second coordination sphere around the catalytic center, which enhanced the activity and stability of two out of the four water oxidation catalysts in aqueous solution, modifying their pH dependence and suppressing the need for adding any organic solvents in solution. Altogether, these results demonstrate how useful artificial metalloenzymes can be for the making of artificial photosynthetic systems

Ultrafast Proton Shuttling in <i>Psammocora</i> Cyan Fluorescent Protein

Cyan, green, yellow, and red fluorescent proteins (FPs) homologous to green fluorescent protein (GFP) are used extensively as model systems to study fundamental processes in photobiology, such as the capture of light energy by protein-embedded chromophores, color tuning by the protein matrix, energy conversion by Förster resonance energy transfer (FRET), and excited-state proton transfer (ESPT) reactions. Recently, a novel cyan fluorescent protein (CFP) termed psamFP488 was isolated from the genus <i>Psammocora</i> of reef building corals. Within the cyan color class, psamFP488 is unusual because it exhibits a significantly extended Stokes shift. Here, we applied ultrafast transient absorption and pump–dump–probe spectroscopy to investigate the mechanistic basis of psamFP488 fluorescence, complemented with fluorescence quantum yield and dynamic light scattering measurements. Transient absorption spectroscopy indicated that, upon excitation at 410 nm, the stimulated cyan emission rises in 170 fs. With pump–dump–probe spectroscopy, we observe a very short-lived (110 fs) ground-state intermediate that we assign to the deprotonated, anionic chromophore. In addition, a minor fraction (14%) decays with 3.5 ps to the ground state. Structural analysis of homologous proteins indicates that Glu-167 is likely positioned in sufficiently close vicinity to the chromophore to act as a proton acceptor. Our findings support a model where unusually fast ESPT from the neutral chromophore to Glu-167 with a time constant of 170 fs and resulting emission from the anionic chromophore forms the basis of the large psamFP488 Stokes shift. When dumped to the ground state, the proton on neutral Glu is very rapidly shuttled back to the anionic chromophore in 110 fs. Proton shuttling in excited and ground states is a factor of 20–4000 faster than in GFP, which probably results from a favorable hydrogen-bonding geometry between the chromophore phenolic oxygen and the glutamate acceptor, possibly involving a short hydrogen bond. At any time in the reaction, the proton is localized on either the chromophore or Glu-167, which implies that most likely no low-barrier hydrogen bond exists between these molecular groups. This work supports the notion that proton transfer in biological systems, be it in an electronic excited or ground state, can be an intrinsically fast process that occurs on a 100 fs time scale. PsamFP488 represents an attractive model system that poses an ultrafast proton transfer regime in discrete steps. It constitutes a valuable model system in addition to wild type GFP, where proton transfer is relatively slow, and the S65T/H148D GFP mutant, where the effects of low-barrier hydrogen bonds dominate

Structure Determination of a Bio-Inspired Self-Assembled Light-Harvesting Antenna by Solid-State NMR and Molecular Modeling

The molecular stacking of an artificial light-harvesting antenna self-assembled from 3¹-aminofunctionalized zinc-chlorins was determined by solid-state NMR in combination with quantum-chemical and molecular-mechanics modeling. A library of trial molecular stacking arrangements was generated based on available structural data for natural and semisynthetic homologues of the Zn-chlorins. NMR assignments obtained for the monomer in solution were validated for self-assembled aggregates and refined with ¹H–¹³C heteronuclear correlation spectroscopy data collected from samples with ¹³C at natural abundance. Solid-state ring-current shifts for the ¹H provided spatial constraints to determine the molecular overlap. This procedure allows for a discrimination between different self-assembled structures and a classification of the stacking mode in terms of electric dipole alignment and π–π interactions, parameters that determine the functional properties of light-harvesting assemblies and conducting nanowires. The combination with quantum-mechanical modeling then allowed building a low-resolution packing model in silico from molecular stacks. The method allows for moderate disorder and residual polymorphism at the stack or molecular level and is generally applicable to determine molecular packing structures of aromatic molecules with structural asymmetry, such as is commonly provided by functionalized side chains that serve to tune the self-assembly process