Dynamics of Proton Transfer to Internal Water during the Photosynthetic Oxygen-Evolving Cycle

In photosynthesis, the light-driven oxidation of water is a sustainable process, which converts solar to chemical energy and produces protons and oxygen. To enable biomimetic strategies, the mechanism of photosynthetic oxygen evolution must be elucidated. Here, we provide information concerning a critical step in the oxygen-evolving, or S-state, cycle. During this S₃-to-S₀ transition, oxygen is produced, and substrate water binds to the manganese–calcium catalytic site. Our spectroscopic and H₂¹⁸O labeling experiments show that this S₃-to-S₀ step is associated with the protonation of an internal water cluster in a hydrogen-bonding network, which contains calcium. When compared to the protonated water cluster, formed during a preceding step, the S₁-to-S₂ transition, the S₃-to-S₀ hydronium ion is likely to be coordinated by additional water molecules. This evidence shows that internal water and the hydrogen bonding network act as a transient proton acceptor at multiple points in the oxygen-evolving cycle

Tracking Reactive Water and Hydrogen-Bonding Networks in Photosynthetic Oxygen Evolution

ConspectusIn oxygenic photosynthesis, photosystem II (PSII) converts water to molecular oxygen through four photodriven oxidation events at a Mn₄CaO₅ cluster. A tyrosine, YZ (Y161 in the D1 polypeptide), transfers oxidizing equivalents from an oxidized, primary chlorophyll donor to the metal center. Calcium or its analogue, strontium, is required for activity. The Mn₄CaO₅ cluster and YZ are predicted to be hydrogen bonded in a water-containing network, which involves amide carbonyl groups, amino acid side chains, and water. This hydrogen-bonded network includes amino acid residues in intrinsic and extrinsic subunits. One of the extrinsic subunits, PsbO, is intrinsically disordered. This extensive (35 Å) network may be essential in facilitating proton release from substrate water. While it is known that some proteins employ internal water molecules to catalyze reactions, there are relatively few methods that can be used to study the role of water. In this Account, we review spectroscopic evidence from our group supporting the conclusion that the PSII hydrogen-bonding network is dynamic and that water in the network plays a direct role in catalysis. Two approaches, transient electron paramagnetic resonance (EPR) and reaction-induced FT-IR (RIFT-IR) spectroscopies, were used. The EPR experiments focused on the decay kinetics of YZ• via recombination at 190 K and the solvent isotope, pH, and calcium dependence of these kinetics. The RIFT-IR experiments focused on shifts in amide carbonyl frequencies, induced by photo-oxidation of the metal cluster, and on the isotope-based assignment of bands to internal, small protonated water clusters at 190, 263, and 283 K. To conduct these experiments, PSII was prepared in selected steps along the catalytic pathway, the S_<i>n</i> state cycle (<i>n</i> = 0–4). This cycle ultimately generates oxygen. In the EPR studies, S-state dependent changes were observed in the YZ• lifetime and in its solvent isotope effect. The YZ• lifetime depended on the presence of calcium at pH 7.5, but not at pH 6.0, suggesting a two-donor model for PCET. At pH 6.0 or 7.5, barium and ammonia both slowed the rate of YZ• recombination, consistent with disruption of the hydrogen-bonding network. In the RIFT-IR studies of the S state transitions, infrared bands associated with the transient protonation and deprotonation of internal waters were identified by D₂O and H₂¹⁸O labeling. The infrared bands of these protonated water clusters, W_<i>n</i>⁺ (or <i>n</i>H₂O­(H₃O)⁺, <i>n</i> = 5–6), exhibited flash dependence and were produced during the S₁ to S₂ and S₃ to S₀ transitions. Calcium dependence was observed at pH 7.5, but not at pH 6.0. S-state induced shifts were observed in amide CO frequencies during the S₁ to S₂ transition and attributed to alterations in hydrogen bonding, based on ammonia sensitivity. In addition, isotope editing of the extrinsic subunit, PsbO, established that amide vibrational bands of this lumenal subunit respond to the S state transitions and that PsbO is a structural template for the reaction center. Taken together, these spectroscopic results support the hypothesis that proton transfer networks, extending from YZ to PsbO, play a functional and dynamic role in photosynthetic oxygen evolution

Chloride Maintains a Protonated Internal Water Network in the Photosynthetic Oxygen Evolving Complex

In photosystem II (PSII), water oxidation occurs at a Mn₄CaO₅ cluster and results in production of molecular oxygen. The Mn₄CaO₅ cluster cycles among five oxidation states, called S<i>_n</i> states. As a result, protons are released at the metal cluster and transferred through a 35 Å hydrogen-bonding network to the lumen. At 283 K, an infrared band at 2830 cm^–1 is assigned to an internal solvated hydronium ion via H₂¹⁸O solvent exchange. This result is similar to a previous report at 263 K. Computations on an oxygen evolving complex model predict that chloride can stabilize a hydronium ion on a network of nine water molecules. In this model, a H₃O⁺ stretching mode at 2738 cm^–1 is predicted to shift to higher frequency with bromide and to lower frequency with nitrate substitution. The calculated frequencies were compared to S₂-minus-S₁ reaction-induced Fourier transform infrared spectra acquired from chloride-, bromide-, or nitrate-containing PSII samples, which were active in oxygen evolution. As predicted, the frequency of the 2830 cm^–1 band shifted to higher energy with bromide and to lower energy with nitrate substitution. These results support the conclusion that an internal hydronium ion and chloride play a direct role in an internal proton transfer event during the S₁-to-S₂ transition

First Site-Specific Incorporation of a Noncanonical Amino Acid into the Photosynthetic Oxygen-Evolving Complex

In photosystem II (PSII), water is oxidized at the oxygen-evolving complex. This process occurs through a light-induced cycle that produces oxygen and protons. While coupled proton and electron transfer reactions play an important role in PSII and other proteins, direct detection of internal proton transfer reactions is challenging. Here, we demonstrate that the unnatural amino acid, 7-azatryptophan (7AW), has unique, pH-sensitive vibrational frequencies, which are sensitive markers of proton transfer. The intrinsically disordered, PSII subunit, PsbO, which contains a single W residue (Trp241), was engineered to contain 7AW at position 241. Fluorescence shows that 7AW-241 is buried in a hydrophobic environment. Reconstitution of 7AW(241)­PsbO to PSII had no significant impact on oxygen evolution activity or flash-dependent protein dynamics. We conclude that directed substitution of 7AW into other structural domains is likely to provide a nonperturbative spectroscopic probe, which can be used to define internal proton pathways in PsbO

Designing a New Strategy for the Formation of IL-in-Oil Microemulsions

Due to the increasing applicability of ionic liquids (ILs) as different components of microemulsions (as the polar liquid, the oil phase, and the surfactant), it would be advantageous to devise a strategy by which we can formulate a microemulsion of our own interest. In this paper, we have shown how we can replace water from water-in-oil microemulsions by ILs to produce IL-in-oil microemulsions. We have synthesized AOT-derived surface-active ionic liquids (SAILs) which can be used to produce a large number of IL-in-oil microemulsions. In particular, we have characterized the phase diagram of the [C₄mim]­[BF₄]/[C₄mim]­[AOT]/benzene ternary system at 298 K. We have shown the formation of IL-in-oil microemulsions using the dynamic light scattering (DLS) technique and using methyl orange (MO), betaine 30, and coumarin-480 (C-480) as probe molecules