Electronic structure and energetics of a heterodimeric BChl g'/Chl a' special pair generated by exposure of Heliomicrobium modesticaldum to dioxygen.

Heliobacteria are anoxygenic phototrophs that have a Type I homodimeric reaction center containing bacteriochlorophyll g (BChl g). Previous experimental studies have shown that in the presence of light and dioxygen, BChl g is converted into 81-OH-chlorophyll aF (hereafter Chl aF), with an accompanying loss of light-driven charge separation. These studies suggest that the reaction center only loses the ability to transfer electrons once both BChl g' molecules of the P800 special pair have been converted to Chl aF'. The present work confirms that the partially converted BChl g'/Chl aF' special pair remains functional in samples exposed to dioxygen by demonstrating its presence using hyperfine couplings obtained from Q-band 1H ENDOR, 2D 14N HYSCORE and DFT methods. The DFT calculations of the BChl g'/BChl g' homodimeric primary donor, which are based on the recently published X-ray crystal structure, predict that the unpaired electron spin is equally delocalized over both BChl g' molecules and provide an excellent match to the experimental hyperfine couplings of the anaerobic samples. Exposure to dioxygen leads to substantial changes in the hyperfine interactions, indicative of greater localization of the unpaired electron spin. The measured hyperfine couplings are reproduced in the DFT calculations by replacing one of the BChl g' molecules of the primary donor with a Chl aF' molecule. The calculations reveal that the spin density becomes localized on BChl g' in the heterodimeric primary donor. Time-dependent DFT calculations demonstrate that conversion of either or both of the accessory BChl g molecules and/or one of the BChl g' molecules of P800 to Chl aF' results in minor effects on the energy of the charge-separated states. In contrast, if both of the BChl g' molecules of P800 are converted a large increase in the energy of the charge-separated state occurs. This suggests that the reaction center remains functional when only one half of the dimer is converted, however, conversion of both halves of the P800 dimer leads to loss of function.

Comparison of proton transfer paths to the QA and QB sites of the Rb. sphaeroides photosynthetic reaction centers.

The photosynthetic bacterial reaction centers from purple non-sulfur bacteria use light energy to drive the transfer of electrons from cytochrome c to ubiquinone. Ubiquinone bound in the QA site cycles between quinone, QA, and anionic semiquinone, QA·-, being reduced once and never binding protons. In the QB site, ubiquinone is reduced twice by QA·-, binds two protons and is released into the membrane as the quinol, QH2. The network of hydrogen bonds formed in a molecular dynamics trajectory was drawn to investigate proton transfer pathways from the cytoplasm to each quinone binding site. QA is isolated with no path for protons to enter from the surface. In contrast, there is a complex and tangled network requiring residues and waters that can bring protons to QB. There are three entries from clusters of surface residues centered around HisH126, GluH224, and HisH68. The network is in good agreement with earlier studies, Mutation of key nodes in the network, such as SerL223, were previously shown to slow proton delivery. Mutational studies had also shown that double mutations of residues such as AspM17 and AspL210 along multiple paths in the network presented here slow the reaction, while single mutations do not. Likewise, mutation of both HisH126 and HisH128, which are at the entry to two paths reduce the rate of proton uptake.

Photosystem II oxygen-evolving complex photoassembly displays an inverse H/D solvent isotope effect under chloride-limiting conditions.

Photosystem II (PSII) performs the solar-driven oxidation of water used to fuel oxygenic photosynthesis. The active site of water oxidation is the oxygen-evolving complex (OEC), a Mn4CaO5 cluster. PSII requires degradation of key subunits and reassembly of the OEC as frequently as every 20 to 40 min. The metals for the OEC are assembled within the PSII protein environment via a series of binding events and photochemically induced oxidation events, but the full mechanism is unknown. A role of proton release in this mechanism is suggested here by the observation that the yield of in vitro OEC photoassembly is higher in deuterated water, D2O, compared with H2O when chloride is limiting. In kinetic studies, OEC photoassembly shows a significant lag phase in H2O at limiting chloride concentrations with an apparent H/D solvent isotope effect of 0.14 ± 0.05. The growth phase of OEC photoassembly shows an H/D solvent isotope effect of 1.5 ± 0.2. We analyzed the protonation states of the OEC protein environment using classical Multiconformer Continuum Electrostatics. Combining experiments and simulations leads to a model in which protons are lost from amino acid that will serve as OEC ligands as metals are bound. Chloride and D2O increase the proton affinities of key amino acid residues. These residues tune the binding affinity of Mn2+/3+ and facilitate the deprotonation of water to form a proposed µ-hydroxo bridged Mn2+Mn3+ intermediate.

Identification of a Na+-Binding Site near the Oxygen-Evolving Complex of Spinach Photosystem II.

The oxygen-evolving complex (OEC) of photosystem II (PSII) is an oxomanganese cluster composed of four redox-active Mn ions and one redox-inactive Ca2+ ion, with two nearby bound Cl- ions. Sodium is a common counterion of both chloride and hydroxide anions, and a sodium-specific binding site has not been identified near the OEC. Here, we find that the oxygen-evolution activity of spinach PSII increases with Na+ concentration, particularly at high pH. A Na+-specific binding site next to the OEC, becomes available after deprotonation of the D1-H337 amino acid residue, is suggested by the analysis of two recently published PSII cryo-electron microscopy maps in combination with quantum mechanical calculations and multiconformation continuum electrostatics simulations.

Relative stability of the S2 isomers of the oxygen evolving complex of photosystem II.

The oxidation of water to O2 is catalyzed by the Oxygen Evolving Complex (OEC), a Mn4CaO5 complex in Photosystem II (PSII). The OEC is sequentially oxidized from state S0 to S4. The S2 state, (MnIII)(MnIV)3, coexists in two redox isomers: S2,g=2, where Mn4 is MnIV and S2,g=4.1, where Mn1 is MnIV. Mn4 has two terminal water ligands, whose proton affinity is affected by the Mn oxidation state. The relative energy of the two S2 redox isomers and the protonation state of the terminal water ligands are analyzed using classical multi-conformer continuum electrostatics (MCCE). The Monte Carlo simulations are done on QM/MM optimized S1 and S2 structures docked back into the complete PSII, keeping the protonation state of the protein at equilibrium with the OEC redox and protonation states. Wild-type PSII, chloride-depleted PSII, PSII in the presence of oxidized YZ/protonated D1-H190, and the PSII mutants D2-K317A, D1-D61A, and D1-S169A are studied at pH 6. The wild-type PSII at pH 8 is also described. In qualitative agreement with experiment, in wild-type PSII, the S2,g=2 redox isomer is the lower energy state; while chloride depletion or pH 8 stabilizes the S2,g=4.1 state and the mutants D2-K317A, D1-D61A, and D1-S169A favor the S2,g=2 state. The protonation states of D1-E329, D1-E65, D1-H337, D1-D61, and the terminal waters on Mn4 (W1 and W2) are affected by the OEC oxidation state. The terminal W2 on Mn4 is a mixture of water and hydroxyl in the S2,g=2 state, indicating the two water protonation states have similar energy, while it remains neutral in the S1 and S2,g=4.1 states. In wild-type PSII, advancement to S2 leads to negligible proton loss and so there is an accumulation of positive charge. In the analyzed mutations and Cl- depleted PSII, additional deprotonation is found upon formation of S2 state.

Thermodynamics of the S2-to-S3 state transition of the oxygen-evolving complex of photosystem II.

The room temperature pump-probe X-ray free electron laser (XFEL) measurements used for serial femtosecond crystallography provide remarkable information about the structures of the catalytic (S-state) intermediates of the oxygen-evolution reaction of photosystem II. However, mixed populations of these intermediates and moderate resolution limit the interpretation of the data from current experiments. The S3 XFEL structures show extra density near the OEC that may correspond to a water/hydroxide molecule. However, in the latest structure, this additional oxygen is 2.08 Šfrom the Oε2 of D1-E189, which is closer than the sum of the van der Waals radii of the two oxygens. Here, we use Boltzmann statistics and Monte Carlo sampling to provide a model for the S2-to-S3 state transition, allowing structural changes and the insertion of an additional water/hydroxide. Based on our model, water/hydroxide addition to the oxygen-evolving complex (OEC) is not thermodynamically favorable in the S2g = 2 state, but it is in the S2g = 4.1 redox isomer. Thus, formation of the S3 state starts by a transition from the S2g = 2 to the S2g = 4.1 structure. Then, electrostatic interactions support protonation of D1-H190 and deprotonation of the Ca2+-ligated water (W3) with proton loss to the lumen. The W3 hydroxide moves toward Mn4, completing the coordination shell of Mn4 and favoring its oxidation to Mn(iv) in the S3 state. In addition, binding an additional hydroxide to Mn1 leads to a conformational change of D1-E189 in the S2g = 4.1 and S3 structures. In the S3 state a fraction of D1-E189 release from Mn1 and bind a proton.

A combined treatment of hydration and dynamical effects for the modeling of host-guest binding thermodynamics: the SAMPL5 blinded challenge.

As part of the SAMPL5 blinded experiment, we computed the absolute binding free energies of 22 host-guest complexes employing a novel approach based on the BEDAM single-decoupling alchemical free energy protocol with parallel replica exchange conformational sampling and the AGBNP2 implicit solvation model specifically customized to treat the effect of water displacement as modeled by the Hydration Site Analysis method with explicit solvation. Initial predictions were affected by the lack of treatment of ionic charge screening, which is very significant for these highly charged hosts, and resulted in poor relative ranking of negatively versus positively charged guests. Binding free energies obtained with Debye-Hückel treatment of salt effects were in good agreement with experimental measurements. Water displacement effects contributed favorably and very significantly to the observed binding affinities; without it, the modeling predictions would have grossly underestimated binding. The work validates the implicit/explicit solvation approach employed here and it shows that comprehensive physical models can be effective at predicting binding affinities of molecular complexes requiring accurate treatment of conformational dynamics and hydration.

Mapping the Oxygens in the Oxygen-Evolving Complex of Photosystem II by Their Nucleophilicity Using Quantum Descriptors.

The oxygen-evolving complex (OEC) of Photosystem II catalyzes the water-splitting reaction using solar energy. Thus, understanding the reaction mechanism will inspire the design of biomimetic artificial catalysts that convert solar energy to chemical energy. Conceptual Density Functional Theory (CDFT) focuses on understanding the reactivity of molecules and the atomic contribution to the overall nucleophilicity and electrophilicity of the molecule using quantum descriptors. However, this method has not been applied to the OEC before. Here, we use Fukui functions and the dual descriptor to provide quantitative measures of the nucleophilicity and electrophilicity of oxygens in the OEC for different models in different S states. Our results show that the µ-oxo bridges connected to terminal Mn4 are nucleophilic, and those in the cube formed by Mn1, Mn2, and Mn3 are mostly electrophilic. The dual descriptors of the bridging oxygens in the OEC showed a similar reactivity to that of bridging oxygens in Mn model compounds. However, the terminal water W1, which is bound to Mn4, showed very strong reactivity in some of the S3 models. Thus, our calculations support the model that proposes the formation of the O2 molecule through nucleophilic attack by a terminal water.

Occupancy Analysis of Water Molecules inside Channels within 25 Å Radius of the Oxygen-Evolving Center of Photosystem II in Molecular Dynamics Simulations.

At room temperature and neutral pH, the oxygen-evolving center (OEC) of photosystem II (PSII) catalyzes water oxidation. During this process, oxygen is released from the OEC, while substrate waters are delivered to the OEC and protons are passed from the OEC to the lumen through water channels known as the narrow or the O4 channel, broad or the Cl1 channel, and large or the O1 channel. Protein residues lining the surfaces of these channels play a critical role in stabilizing the hydrogen-bonding networks that assist in the process. We carried out an occupancy analysis to better understand the structural and possible substrate water dynamics in full PSII monomer molecular dynamics (MD) trajectories in both the S1 and S2 states. We find that the equilibrated positions of water molecules derived from MD-derived electron density maps largely match the experimentally observed positions in crystallography. Furthermore, the occupancy reduction in MD simulations of some water molecules inside the single-filed narrow channel also correlates well with the crystallographic data during a structural transition when the S1 state of the OEC advances to the S2 state. The overall reduced occupancies of water molecules are the source of their "vacancy-hopping" dynamic nature inside these channels, unlike water molecules inside an ice lattice where all water molecules have a fixed unit occupancy. We propose on the basis of findings in our structural and molecular dynamics analysis that the water molecule occupying a pocket formed by D1-D61, D1-S169, and O4 of the OEC could be the last steppingstone to enter into the OEC and that the broad channel may be favored for proton transfer.

Protein Motifs for Proton Transfers That Build the Transmembrane Proton Gradient.

Biological membranes are barriers to polar molecules, so membrane embedded proteins control the transfers between cellular compartments. Protein controlled transport moves substrates and activates cellular signaling cascades. In addition, the electrochemical gradient across mitochondrial, bacterial and chloroplast membranes, is a key source of stored cellular energy. This is generated by electron, proton and ion transfers through proteins. The gradient is used to fuel ATP synthesis and to drive active transport. Here the mechanisms by which protons move into the buried active sites of Photosystem II (PSII), bacterial RCs (bRCs) and through the proton pumps, Bacteriorhodopsin (bR), Complex I and Cytochrome c oxidase (CcO), are reviewed. These proteins all use water filled proton transfer paths. The proton pumps, that move protons uphill from low to high concentration compartments, also utilize Proton Loading Sites (PLS), that transiently load and unload protons and gates, which block backflow of protons. PLS and gates should be synchronized so PLS proton affinity is high when the gate opens to the side with few protons and low when the path is open to the high concentration side. Proton transfer paths in the proteins we describe have different design features. Linear paths are seen with a unique entry and exit and a relatively straight path between them. Alternatively, paths can be complex with a tangle of possible routes. Likewise, PLS can be a single residue that changes protonation state or a cluster of residues with multiple charge and tautomer states.

Proton exit pathways surrounding the oxygen evolving complex of photosystem II.

Photosystem II allows water to be the primary electron source for the photosynthetic electron transfer chain. Water is oxidized to dioxygen at the Oxygen Evolving Complex (OEC), a Mn4CaO5 inorganic core embedded on the lumenal side of PSII. Water-filled channels surrounding the OEC must bring in substrate water molecules, remove the product protons to the lumen, and may transport the product oxygen. Three water-filled channels, denoted large, narrow, and broad, extend from the OEC towards the aqueous surface more than 15 Å away. However, the role of each pathway in the transport in and out of the OEC is yet to be established. Here, we combine Molecular Dynamics (MD), Multi Conformation Continuum Electrostatics (MCCE) and Network Analysis to compare and contrast the three potential proton transfer paths. Hydrogen bond network analysis shows that near the OEC the waters are highly interconnected with similar free energy for hydronium at all locations. The paths diverge as they move towards the lumen. The water chain in the broad channel is better connected than in the narrow and large channels, where disruptions in the network are observed approximately 10 Å from the OEC. In addition, the barrier for hydronium translocation is lower in the broad channel. Thus, a proton released from any location on the OEC can access all paths, but the likely exit to the lumen passes through PsbO via the broad channel.

Hydrogen bond network analysis reveals the pathway for the proton transfer in the E-channel of T. thermophilus Complex I.

Complex I, NADH-ubiquinone oxidoreductase, is the first enzyme in the mitochondrial and bacterial aerobic respiratory chain. It pumps four protons through four transiently open pathways from the high pH, negative, N-side of the membrane to the positive, P-side driven by the exergonic transfer of electrons from NADH to a quinone. Three protons transfer through subunits descended from antiporters, while the fourth, E-channel is unique. The path through the E-channel is determined by a network analysis of hydrogen bonded pathways obtained by Monte Carlo sampling of protonation states, polar hydrogen orientation and water occupancy. Input coordinates are derived from molecular dynamics trajectories comparing oxidized, reduced (dihydro) and no menaquinone-8 (MQ). A complex proton transfer path from the N- to the P-side is found consisting of six clusters of highly connected hydrogen-bonded residues. The network connectivity depends on the presence of quinone and its redox state, supporting a role for this cofactor in coupling electron and proton transfers. The N-side is more organized with MQ-bound complex I facilitating proton entry, while the P-side is more connected in the apo-protein, facilitating proton exit. Subunit Nqo8 forms the core of the E channel; Nqo4 provides the N-side entry, Nqo7 and then Nqo10 join the pathway in the middle, while Nqo11 contributes to the P-side exit.

Identifying the proton loading site cluster in the ba3 cytochrome c oxidase that loads and traps protons.

Cytochrome c Oxidase (CcO) is the terminal electron acceptor in aerobic respiratory chain, reducing O2 to water. The released free energy is stored by pumping protons through the protein, maintaining the transmembrane electrochemical gradient. Protons are held transiently in a proton loading site (PLS) that binds and releases protons driven by the electron transfer reaction cycle. Multi-Conformation Continuum Electrostatics (MCCE) was applied to crystal structures and Molecular Dynamics snapshots of the B-type Thermus thermophilus CcO. Six residues are identified as the PLS, binding and releasing protons as the charges on heme b and the binuclear center are changed: the heme a3 propionic acids, Asp287, Asp372, His376 and Glu126B. The unloaded state has one proton and the loaded state two protons on these six residues. Different input structures, modifying the PLS conformation, show different proton distributions and result in different proton pumping behaviors. One loaded and one unloaded protonation states have the loaded/unloaded states close in energy so the PLS binds and releases a proton through the reaction cycle. The alternative proton distributions have state energies too far apart to be shifted by the electron transfers so are locked in loaded or unloaded states. Here the protein can use active states to load and unload protons, but has nearby trapped states, which stabilize PLS protonation state, providing new ideas about the CcO proton pumping mechanism. The distance between the PLS residues Asp287 and His376 correlates with the energy difference between loaded and unloaded states.