High-resolution cryo-electron microscopy structure of photosystem II from the mesophilic cyanobacterium, Synechocystis sp. PCC 6803.

Photosystem II (PSII) enables global-scale, light-driven water oxidation. Genetic manipulation of PSII from the mesophilic cyanobacterium Synechocystis sp. PCC 6803 has provided insights into the mechanism of water oxidation; however, the lack of a high-resolution structure of oxygen-evolving PSII from this organism has limited the interpretation of biophysical data to models based on structures of thermophilic cyanobacterial PSII. Here, we report the cryo-electron microscopy structure of PSII from Synechocystis sp. PCC 6803 at 1.93-Å resolution. A number of differences are observed relative to thermophilic PSII structures, including the following: the extrinsic subunit PsbQ is maintained, the C terminus of the D1 subunit is flexible, some waters near the active site are partially occupied, and differences in the PsbV subunit block the Large (O1) water channel. These features strongly influence the structural picture of PSII, especially as it pertains to the mechanism of water oxidation.

Tools for analyzing protonation states and for tracing proton transfer pathways with examples from the Rb. sphaeroides photosynthetic reaction centers.

Protons participate in many reactions. In proteins, protons need paths to move in and out of buried active sites. The vectorial movement of protons coupled to electron transfer reactions establishes the transmembrane electrochemical gradient used for many reactions, including ATP synthesis. Protons move through hydrogen bonded chains of waters and hydroxy side chains via the Grotthuss mechanism and by proton binding and release from acidic and basic residues. MCCE analysis shows that proteins exist in a large number of protonation states. Knowledge of the equilibrium ensemble can provide a rational basis for setting protonation states in simulations that fix them, such as molecular dynamics (MD). The proton path into the QB site in the bacterial reaction centers (RCs) of Rb. sphaeroides is analyzed by MD to provide an example of the benefits of using protonation states found by the MCCE program. A tangled web of side chains and waters link the cytoplasm to QB. MCCE analysis of snapshots from multiple trajectories shows that changing the input protonation state of a residue in MD biases the trajectory shifting the proton affinity of that residue. However, the proton affinity of some residues is more sensitive to the input structure. The proton transfer networks derived from different trajectories are quite robust. There are some changes in connectivity that are largely restricted to the specific residues whose protonation state is changed. Trajectories with QB•- are compared with earlier results obtained with QB [Wei et. al Photosynthesis Research volume 152, pages153-165 (2022)] showing only modest changes. While introducing new methods the study highlights the difficulty of establishing the connections between protein conformation.

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.

Overview of the SAMPL6 pKa challenge: evaluating small molecule microscopic and macroscopic pKa predictions.

The prediction of acid dissociation constants (pKa) is a prerequisite for predicting many other properties of a small molecule, such as its protein-ligand binding affinity, distribution coefficient (log D), membrane permeability, and solubility. The prediction of each of these properties requires knowledge of the relevant protonation states and solution free energy penalties of each state. The SAMPL6 pKa Challenge was the first time that a separate challenge was conducted for evaluating pKa predictions as part of the Statistical Assessment of Modeling of Proteins and Ligands (SAMPL) exercises. This challenge was motivated by significant inaccuracies observed in prior physical property prediction challenges, such as the SAMPL5 log D Challenge, caused by protonation state and pKa prediction issues. The goal of the pKa challenge was to assess the performance of contemporary pKa prediction methods for drug-like molecules. The challenge set was composed of 24 small molecules that resembled fragments of kinase inhibitors, a number of which were multiprotic. Eleven research groups contributed blind predictions for a total of 37 pKa distinct prediction methods. In addition to blinded submissions, four widely used pKa prediction methods were included in the analysis as reference methods. Collecting both microscopic and macroscopic pKa predictions allowed in-depth evaluation of pKa prediction performance. This article highlights deficiencies of typical pKa prediction evaluation approaches when the distinction between microscopic and macroscopic pKas is ignored; in particular, we suggest more stringent evaluation criteria for microscopic and macroscopic pKa predictions guided by the available experimental data. Top-performing submissions for macroscopic pKa predictions achieved RMSE of 0.7-1.0 pKa units and included both quantum chemical and empirical approaches, where the total number of extra or missing macroscopic pKas predicted by these submissions were fewer than 8 for 24 molecules. A large number of submissions had RMSE spanning 1-3 pKa units. Molecules with sulfur-containing heterocycles or iodo and bromo groups were less accurately predicted on average considering all methods evaluated. For a subset of molecules, we utilized experimentally-determined microstates based on NMR to evaluate the dominant tautomer predictions for each macroscopic state. Prediction of dominant tautomers was a major source of error for microscopic pKa predictions, especially errors in charged tautomers. The degree of inaccuracy in pKa predictions observed in this challenge is detrimental to the protein-ligand binding affinity predictions due to errors in dominant protonation state predictions and the calculation of free energy corrections for multiple protonation states. Underestimation of ligand pKa by 1 unit can lead to errors in binding free energy errors up to 1.2 kcal/mol. The SAMPL6 pKa Challenge demonstrated the need for improving pKa prediction methods for drug-like molecules, especially for challenging moieties and multiprotic molecules.

Evaluation of log P, pKa, and log D predictions from the SAMPL7 blind challenge.

The Statistical Assessment of Modeling of Proteins and Ligands (SAMPL) challenges focuses the computational modeling community on areas in need of improvement for rational drug design. The SAMPL7 physical property challenge dealt with prediction of octanol-water partition coefficients and pKa for 22 compounds. The dataset was composed of a series of N-acylsulfonamides and related bioisosteres. 17 research groups participated in the log P challenge, submitting 33 blind submissions total. For the pKa challenge, 7 different groups participated, submitting 9 blind submissions in total. Overall, the accuracy of octanol-water log P predictions in the SAMPL7 challenge was lower than octanol-water log P predictions in SAMPL6, likely due to a more diverse dataset. Compared to the SAMPL6 pKa challenge, accuracy remains unchanged in SAMPL7. Interestingly, here, though macroscopic pKa values were often predicted with reasonable accuracy, there was dramatically more disagreement among participants as to which microscopic transitions produced these values (with methods often disagreeing even as to the sign of the free energy change associated with certain transitions), indicating far more work needs to be done on pKa prediction methods.

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.

Mesoscopic to Macroscopic Electron Transfer by Hopping in a Crystal Network of Cytochromes.

Rapid and directed electron transfer (ET) is essential for biological processes. While the rates of ET over 1-2 nm in proteins can largely be described by simplified nonadiabatic theory, it is not known how these processes scale to microscopic distances. We generated crystalline lattices of Small Tetraheme Cytochromes (STC) forming well-defined, three-dimensional networks of closely spaced redox centers that appear to be nearly ideal for multistep ET. Electrons were injected into specific locations in the STC crystals by direct photoreduction, and their redistribution was monitored by imaging. The results demonstrate ET over mesoscopic to microscopic (∼100 µm) distances through sequential hopping in a biologically based heme network. We estimate that a hypothetical "nanowire" composed of crystalline STC with a cross-section of about 100 cytochromes could support the anaerobic respiration of a Shewanella cell. The crystalline lattice insulates mobile electrons from oxidation by O2, as compared to those in cytochromes in solution, potentially allowing for efficient delivery of current without production of reactive oxygen species. The platform allows direct tests of whether the assumptions based on short-range ET hold for sequential ET over mesoscopic distances. We estimate that the interprotein ET across 6 Å between hemes in adjacent proteins was about 105 s-1, about 100-fold slower than expectations based on simplified theory. More detailed analyses implied that additional factors, possibly contributed by the crystal lattice, may strongly impact mesoscale ET mainly by increasing the reorganizational energy of interprotein ET, which suggests design strategies for engineering improved nanowires suitable for future bioelectronic materials.

Charge Transfer and Chemo-Mechanical Coupling in Respiratory Complex I.

The mitochondrial respiratory chain, formed by five protein complexes, utilizes energy from catabolic processes to synthesize ATP. Complex I, the first and the largest protein complex of the chain, harvests electrons from NADH to reduce quinone, while pumping protons across the mitochondrial membrane. Detailed knowledge of the working principle of such coupled charge-transfer processes remains, however, fragmentary due to bottlenecks in understanding redox-driven conformational transitions and their interplay with the hydrated proton pathways. Complex I from Thermus thermophilus encases 16 subunits with nine iron-sulfur clusters, reduced by electrons from NADH. Here, employing the latest crystal structure of T. thermophilus complex I, we have used microsecond-scale molecular dynamics simulations to study the chemo-mechanical coupling between redox changes of the iron-sulfur clusters and conformational transitions across complex I. First, we identify the redox switches within complex I, which allosterically couple the dynamics of the quinone binding pocket to the site of NADH reduction. Second, our free-energy calculations reveal that the affinity of the quinone, specifically menaquinone, for the binding-site is higher than that of its reduced, menaquinol form-a design essential for menaquinol release. Remarkably, the barriers to diffusive menaquinone dynamics are lesser than that of the more ubiquitous ubiquinone, and the naphthoquinone headgroup of the former furnishes stronger binding interactions with the pocket, favoring menaquinone for charge transport in T. thermophilus. Our computations are consistent with experimentally validated mutations and hierarchize the key residues into three functional classes, identifying new mutation targets. Third, long-range hydrogen-bond networks connecting the quinone-binding site to the transmembrane subunits are found to be responsible for proton pumping. Put together, the simulations reveal the molecular design principles linking redox reactions to quinone turnover to proton translocation in complex I.

Standard state free energies, not pKas, are ideal for describing small molecule protonation and tautomeric states.

The pKa is the standard measure used to describe the aqueous proton affinity of a compound, indicating the proton concentration (pH) at which two protonation states (e.g. A- and AH) have equal free energy. However, compounds can have additional protonation states (e.g. AH2+), and may assume multiple tautomeric forms, with the protons in different positions (microstates). Macroscopic pKas give the pH where the molecule changes its total number of protons, while microscopic pKas identify the tautomeric states involved. As tautomers have the same number of protons, the free energy difference between them and their relative probability is pH independent so there is no pKa connecting them. The question arises: What is the best way to describe protonation equilibria of a complex molecule in any pH range? Knowing the number of protons and the relative free energy of all microstates at a single pH, ∆G°, provides all the information needed to determine the free energy, and thus the probability of each microstate at each pH. Microstate probabilities as a function of pH generate titration curves that highlight the low energy, observable microstates, which can then be compared with experiment. A network description connecting microstates as nodes makes it straightforward to test thermodynamic consistency of microstate free energies. The utility of this analysis is illustrated by a description of one molecule from the SAMPL6 Blind pKa Prediction Challenge. Analysis of microstate ∆G°s also makes a more compact way to archive and compare the pH dependent behavior of compounds with multiple protonatable sites.

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.

L-2-Hydroxyglutarate production arises from noncanonical enzyme function at acidic pH.

The metabolite 2-hydroxyglutarate (2HG) can be produced as either a D-R- or L-S- enantiomer, each of which inhibits α-ketoglutarate (αKG)-dependent enzymes involved in diverse biologic processes. Oncogenic mutations in isocitrate dehydrogenase (IDH) produce D-2HG, which causes a pathologic blockade in cell differentiation. On the other hand, oxygen limitation leads to accumulation of L-2HG, which can facilitate physiologic adaptation to hypoxic stress in both normal and malignant cells. Here we demonstrate that purified lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) catalyze stereospecific production of L-2HG via 'promiscuous' reduction of the alternative substrate αKG. Acidic pH enhances production of L-2HG by promoting a protonated form of αKG that binds to a key residue in the substrate-binding pocket of LDHA. Acid-enhanced production of L-2HG leads to stabilization of hypoxia-inducible factor 1 alpha (HIF-1α) in normoxia. These findings offer insights into mechanisms whereby microenvironmental factors influence production of metabolites that alter cell fate and function.

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.

Two Cl Ions and a Glu Compete for a Helix Cage in the CLC Proton/Cl- Antiporter.

The ubiquitously expressed CLC chloride transporters are involved in a great variety of physiological functions. The CLC protein fold is shared by Cl- channels and 2Cl-:1H+ antiporters. The antiporters pump three charges per cycle across the membrane with two Cl ions moving in the opposite direction of one proton. Multiconformational continuum electrostatics was used to calculate the coupled thermodynamics of the protonation of the extracellular-facing gating Glu (Ex) and Cl- binding to the external (Sx) and central (Sc) sites in CLC-ec1, the Escherichia coli exchanger. Sx, Sc, and Ex are buried within the protein where the intersection of two helix N-termini creates a region with a strong, localized positive potential for anion binding. Our chemical potential titrations describe the thermodynamic linkage for binding the Cl- to each site and protons to Ex. We find that the 2Cl-:1H+ binding stoichiometry is a result of Cl- binding to Sx requiring H+ binding to Ex, whereas Cl- binding to Sc does not lead to proton uptake. When Sx binds a Cl-, the protonated Ex moves upward, out of the positive helix cage. The increasing Ex proton affinity on binding the first Cl- reduces the cost of binding the second Cl- at either Sx or Sc. Despite the repulsion among the anions, the lowest energy states have two anions bound in the helix cage. The state with no Cl- is not favored electrostatically, but relies on Ex blocking Sx and on the central residues Y445 and S107 blocking Sc.

The design features cells use to build their transmembrane proton gradient.

Organisms store energy from food and sunlight as an electrochemical gradient across the membranes of mitochondria, chloroplasts and bacteria. The gradient arises from differences in the concentration of protons and ions on the negative (N) and positive (P) sides of these membranes. This perspective describes how the proton gradient is formed. One strategy is the movement of electrons but not protons across a membrane-embedded protein from a site of proton-releasing oxidative chemistry on the P-side of the protein to a site of proton-binding reductive chemistry on the N-side. Alternately, protons are directly pumped across membrane-embedded proteins, which have gated proton transfer pathways that are opened and closed, as well as internal sites where the proton affinity varies as the protein goes through the reaction cycle. The molecules that carry out these roles are complex, utilizing non-amino acid cofactors and earth-abundant metals. However, these are also potential sources of high-energy toxic byproducts. Understanding these reactions can open the door to their rational redesign, with possible beneficial effects as far-reaching as improving the global food supply, preventing neurodegenerative diseases, and better understanding the role of metabolism in aging.

Halorhodopsin pumps Cl- and bacteriorhodopsin pumps protons by a common mechanism that uses conserved electrostatic interactions.

Key mutations differentiate the functions of homologous proteins. One example compares the inward ion pump halorhodopsin (HR) and the outward proton pump bacteriorhodopsin (BR). Of the nine essential buried ionizable residues in BR, six are conserved in HR. However, HR changes three BR acids, D85 in a central cluster of ionizable residues, D96, nearer the intracellular, and E204, nearer the extracellular side of the membrane to the small, neutral amino acids T111, V122, and T230, respectively. In BR, acidic amino acids are stationary anions whose proton affinity is modulated by conformational changes, establishing a sequence of directed binding and release of protons. Multiconformation continuum electrostatics calculations of chloride affinity and residue protonation show that, in reaction intermediates where an acid is ionized in BR, a Cl(-) is bound to HR in a position near the deleted acid. In the HR ground state, Cl(-) binds tightly to the central cluster T111 site and weakly to the extracellular T230 site, recovering the charges on ionized BR-D85 and neutral E204 in BR. Imposing key conformational changes from the BR M intermediate into the HR structure results in the loss of Cl(-) from the central T111 site and the tight binding of Cl(-) to the extracellular T230 site, mirroring the changes that protonate BR-D85 and ionize E204 in BR. The use of a mobile chloride in place of D85 and E204 makes HR more susceptible to the environmental pH and salt concentrations than BR. These studies shed light on how ion transfer mechanisms are controlled through the interplay of protein and ion electrostatics.

Characterizing the proton loading site in cytochrome c oxidase.

Cytochrome c oxidase (CcO) uses the energy released by reduction of O2 to H2O to drive eight charges from the high pH to low pH side of the membrane, increasing the electrochemical gradient. Four electrons and protons are used for chemistry, while four more protons are pumped. Proton pumping requires that residues on a pathway change proton affinity through the reaction cycle to load and then release protons. The protonation states of all residues in CcO are determined in MultiConformational Continuum Electrostatics simulations with the protonation and redox states of heme a, a3, Cu(B), Y288, and E286 used to define the catalytic cycle. One proton is found to be loaded and released from residues identified as the proton loading site (PLS) on the P-side of the protein in each of the four CcO redox states. Thus, the same proton pumping mechanism can be used each time CcO is reduced. Calculations with structures of Rhodobacter sphaeroides, Paracoccus denitrificans, and bovine CcO derived by crystallography and molecular dynamics show the PLS functions similarly in different CcO species. The PLS is a cluster rather than a single residue, as different structures show 1-4 residues load and release protons. However, the proton affinity of the heme a3 propionic acids primarily determines the number of protons loaded into the PLS; if their proton affinity is too low, less than one proton is loaded.

Unraveling the mechanism of proton translocation in the extracellular half-channel of bacteriorhodopsin.

Bacteriorhodopsin, a light activated protein that creates a proton gradient in halobacteria, has long served as a simple model of proton pumps. Within bacteriorhodopsin, several key sites undergo protonation changes during the photocycle, moving protons from the higher pH cytoplasm to the lower pH extracellular side. The mechanism underlying the long-range proton translocation between the central (the retinal Schiff base SB216, D85, and D212) and exit clusters (E194 and E204) remains elusive. To obtain a dynamic view of the key factors controlling proton translocation, a systematic study using molecular dynamics simulation was performed for eight bacteriorhodopsin models varying in retinal isomer and protonation states of the SB216, D85, D212, and E204. The side-chain orientation of R82 is determined primarily by the protonation states of the residues in the EC. The side-chain reorientation of R82 modulates the hydrogen-bond network and consequently possible pathways of proton transfer. Quantum mechanical intrinsic reaction coordinate calculations of proton-transfer in the methyl guanidinium-hydronium-hydroxide model system show that proton transfer via a guanidinium group requires an initial geometry permitting proton donation and acceptance by the same amine. In all the bacteriorhodopsin models, R82 can form proton wires with both the CC and the EC connected by the same amine. Alternatively, rare proton wires for proton transfer from the CC to the EC without involving R82 were found in an O' state where the proton on D85 is transferred to D212.

Changing hydration level in an internal cavity modulates the proton affinity of a key glutamate in cytochrome c oxidase.

Cytochrome c oxidase contributes to the transmembrane proton gradient by removing two protons from the high-pH side of the membrane each time the binuclear center active site is reduced. One proton goes to the binuclear center, whereas the other is pumped to the low-pH periplasmic space. Glutamate 286 (Glu286) has been proposed to serve as a transiently deprotonated proton donor. Using unrestrained atomistic molecular dynamics simulations, we show that the size of and water distribution in the hydrophobic cavity that holds Glu286 is controlled by the protonation state of the propionic acid of heme a3, a group on the proton outlet pathway. Protonation of the propionate disrupts hydrogen bonding to two side chains, allowing a loop to swing open. Continuum electrostatics and atomistic free-energy perturbation calculations show that the resultant changes in hydration and electrostatic interactions lower the Glu proton affinity by at least 5 kcal/mol. These changes in the internal hydration level occur in the absence of major conformational transitions and serve to stabilize needed transient intermediates in proton transport. The trigger is not the protonation of the Glu of interest, but rather the protonation of a residue ∼10 Šaway. Thus, unlike local water penetration to stabilize a new charge, this finding represents a specific role for water molecules in the protein interior, mediating proton transfers and facilitating ion transport.

Molecular mechanisms for generating transmembrane proton gradients.

Membrane proteins use the energy of light or high energy substrates to build a transmembrane proton gradient through a series of reactions leading to proton release into the lower pH compartment (P-side) and proton uptake from the higher pH compartment (N-side). This review considers how the proton affinity of the substrates, cofactors and amino acids are modified in four proteins to drive proton transfers. Bacterial reaction centers (RCs) and photosystem II (PSII) carry out redox chemistry with the species to be oxidized on the P-side while reduction occurs on the N-side of the membrane. Terminal redox cofactors are used which have pKas that are strongly dependent on their redox state, so that protons are lost on oxidation and gained on reduction. Bacteriorhodopsin is a true proton pump. Light activation triggers trans to cis isomerization of a bound retinal. Strong electrostatic interactions within clusters of amino acids are modified by the conformational changes initiated by retinal motion leading to changes in proton affinity, driving transmembrane proton transfer. Cytochrome c oxidase (CcO) catalyzes the reduction of O2 to water. The protons needed for chemistry are bound from the N-side. The reduction chemistry also drives proton pumping from N- to P-side. Overall, in CcO the uptake of 4 electrons to reduce O2 transports 8 charges across the membrane, with each reduction fully coupled to removal of two protons from the N-side, the delivery of one for chemistry and transport of the other to the P-side.