Protonation-State-Dependent Communication in Cytochrome c Oxidase.

Proton transfer in cytochrome c oxidase from the cellular inside to the binuclear redox center (BNC) can occur through two distinct pathways, the D- and K-channels. For the protein to function as both a redox enzyme and a proton pump, proton transfer into the protein toward the BNC or toward a proton loading site (and ultimately through the membrane) must be highly regulated. The PR → F transition is the first step in a catalytic cycle that requires proton transfer from the bulk at the N-side to the BNC. Molecular dynamics simulations of the PR → F intermediate of this transition, with 16 different combinations of protonation states of key residues in the D- and K-channel, show the impact of the K-channel on the D-channel to be protonation-state dependent. Strength as well as means of communication, correlations in positions, or communication along the hydrogen-bonded network depends on the protonation state of the K-channel residue K362. The conformational and hydrogen-bond dynamics of the D-channel residue N139 is regulated by an interplay of protonation in the D-channel and K362. N139 thus assumes a gating function by which proton passage through the D-channel toward E286 is likely facilitated for states with protonated K362 and unprotonated E286. In contrast, proton passage through the D-channel is hindered by N139's preference for a closed conformation in situations with protonated E286.

Redox induced protonation of heme propionates in cytochrome c oxidase: Insights from surface enhanced resonance Raman spectroscopy and QM/MM calculations.

Understanding the coupling between heme reduction and proton translocation in cytochrome c oxidase (CcO) is still an open problem. The propionic acids of heme a3 have been proposed to act as a proton loading site (PLS) in the proton pumping pathway, yet this proposal could not be verified by experimental data so far. We have set up an experiment where the redox states of the two hemes in CcO can be controlled via external electrical potential. Surface enhanced resonance Raman (SERR) spectroscopy was applied to simultaneously monitor the redox state of the hemes and the protonation state of the heme propionates. Simulated spectra based on QM/MM calculations were used to assign the resonant enhanced CH2 twisting modes of the propionates to the protonation state of the individual heme a and heme a3 propionates respectively. The comparison between calculated and measured H2OD2O difference spectra allowed a sound band assignment. In the fully reduced enzyme at least three of the four heme propionates were found to be protonated whereas in the presence of a reduced heme a and an oxidized heme a3 only protonation of one heme a3 propionates was observed. Our data supports the postulated scenario where the heme a3 propionates are involved in the proton pathway.

The reductive phase of Rhodobacter sphaeroides cytochrome c oxidase disentangled by CO ligation.

Cytochrome c oxidase (CcO) is a membrane protein of the respiratory chain that catalytically reduces molecular oxygen (O2) to water while translocating protons across the membrane. The enzyme hosts two copper and two heme iron moieties (heme a/heme a3). The atomic details of the sequential steps that go along with this redox-driven proton translocation are a matter of debate. Particularly for the reductive phase of CcO that precedes oxygen binding experimental data are scarce. Here, we use CcO under anaerobic conditions where carbon monoxide (CO) is bound to heme a3 which in tandem with CuB forms the binuclear center (BNC). Fourier-transform infrared (FTIR) absorption spectroscopy is combined with electro-chemistry to probe different redox and protonation states populated by variation of the external electrostatic potential. With this approach, the redox behavior of heme a and the BNC could be separated and the corresponding redox potentials were determined. We also infer the protonation of one of the propionate side chains of heme a3 to correlate with the oxidation of heme a. Experimental changes in the local electric field surrounding CO bound to heme a3 are determined by their vibrational Stark effect and agree well with electrostatic computations. The comparison of experimental and computational results indicates that changes of the heme a3/CuB redox state are coupled to proton transfer towards heme a3. The latter supports the role of the heme a3 propionate D as proton loading site.

Retraction: The reductive phase of Rhodobacter sphaeroides cytochrome c oxidase disentangled by CO ligation.

Retraction of 'The reductive phase of Rhodobacter sphaeroides cytochrome c oxidase disentangled by CO ligation' by Hendrik Mohrmann et al., Phys. Chem. Chem. Phys., 2017, DOI: .

RETRACTED: Protonation State-Dependent Communication in Cytochrome c Oxidase.

Proton transfer in cytochrome c oxidase from the cellular inside to the binuclear redox center (BNC) can occur through two distinct pathways, the D- and K-channels. For the protein to function as both redox enzyme and proton pump, proton transfer out of either of the channels toward the BNC or into the protein toward a proton loading site, and ultimately through the membrane, must be highly regulated. The O→E intermediate of cytochrome c oxidase is the first redox state in its catalytic cycle, where proton transfer through the K-channel, from K362 to Y288 at the BNC, is important. Molecular dynamics simulations of this intermediate with 16 different combinations of protonation states of key residues in the D- and K-channel show the mutual impact of the two proton-conducting channels to be protonation state-dependent. Strength as well as means of communication, correlations in positions, or connections along the hydrogen-bonded network, change with the protonation state of the K-channel residue K362. The conformational and hydrogen-bond dynamics of the D-channel residue N139 regulated by an interplay of protonation in the D-channel and K362. N139 thus assumes a gating function by which proton passage through the D-channel toward E286 is likely facilitated for states with protonated K362 and unprotonated E286, which would in principle allow proton transfer to the BNC, but no proton pumping until a proton has reached E286.

Proton solvation in protic and aprotic solvents.

Protonation pattern strongly affects the properties of molecular systems. To determine protonation equilibria, proton solvation free energy, which is a central quantity in solution chemistry, needs to be known. In this study, proton affinities (PAs), electrostatic energies of solvation, and pKA values were computed in protic and aprotic solvents. The proton solvation energy in acetonitrile (MeCN), methanol (MeOH), water, and dimethyl sulfoxide (DMSO) was determined from computed and measured pKA values for a specially selected set of organic compounds. pKA values were computed with high accuracy using a combination of quantum chemical and electrostatic approaches. Quantum chemical density functional theory computations were performed evaluating PA in the gas-phase. The electrostatic contributions of solvation were computed solving the Poisson equation. The computations yield proton solvation free energies with high accuracy, which are in MeCN, MeOH, water, and DMSO -255.1, -265.9, -266.3, and -266.4 kcal/mol, respectively, where the value for water is close to the consensus value of -265.9 kcal/mol. The pKA values of MeCN, MeOH, and DMSO in water correlates well with the corresponding proton solvation energies in these liquids, indicating that the solvated proton was attached to a single solvent molecule.

Lysine 362 in cytochrome c oxidase regulates opening of the K-channel via changes in pKA and conformation.

The metabolism of aerobic life uses the conversion of molecular oxygen to water as an energy source. This reaction is catalyzed by cytochrome e oxidase (CeO) consuming four electrons and four protons, which move along specific routes. While all four electrons are transferred via the same cofactors to the binuclear reaction center (BNC), the protons take two different routes in the A-type CeO, i.e., two of the four chemical protons consumed in the reaction arrive via the D-channel in the oxidative first half starting after oxygen binding. The other two chemical protons enter via the K-channel in the reductive second half of the reaction cycle. To date, the mechanism behind these separate proton transport pathways has not been understood. In this study, we propose a model that can explain the reaction-step specific opening and closing of the K-channel by conformational and pKA changes of its central lysine 362. Molecular dynamics simulations reveal an upward movement of Lys362 towards the BNC, which had already been supposed by several experimental studies. Redox state-dependent pKA calculations provide evidence that Lys362 may protonate transiently, thereby opening the K-channel only in the reductive second half of the reaction cycle. From our results, we develop a model that assigns a key role to Lys362 in the proton gating between the two proton input channels of the A-type CeO.

Reprint of PSII manganese cluster: protonation of W2, O5, O4 and His337 in the S1 state explored by combined quantum chemical and electrostatic energy computations.

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn4CaO5-cluster (Mn-cluster) in four discrete oxidation steps [S1-(S4/S0)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.

PSII manganese cluster: protonation of W2, O5, O4 and His337 in the S1 state explored by combined quantum chemical and electrostatic energy computations.

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn4CaO5-cluster (Mn-cluster) in four discrete oxidation steps [S1-(S4/S0)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.

Optimized distance-dependent atom-pair-based potential DOOP for protein structure prediction.

The DOcking decoy-based Optimized Potential (DOOP) energy function for protein structure prediction is based on empirical distance-dependent atom-pair interactions. To optimize the atom-pair interactions, native protein structures are decomposed into polypeptide chain segments that correspond to structural motives involving complete secondary structure elements. They constitute near native ligand-receptor systems (or just pairs). Thus, a total of 8609 ligand-receptor systems were prepared from 954 selected proteins. For each of these hypothetical ligand-receptor systems, 1000 evenly sampled docking decoys with 0-10 Å interface root-mean-square-deviation (iRMSD) were generated with a method used before for protein-protein docking. A neural network-based optimization method was applied to derive the optimized energy parameters using these decoys so that the energy function mimics the funnel-like energy landscape for the interaction between these hypothetical ligand-receptor systems. Thus, our method hierarchically models the overall funnel-like energy landscape of native protein structures. The resulting energy function was tested on several commonly used decoy sets for native protein structure recognition and compared with other statistical potentials. In combination with a torsion potential term which describes the local conformational preference, the atom-pair-based potential outperforms other reported statistical energy functions in correct ranking of native protein structures for a variety of decoy sets. This is especially the case for the most challenging ROSETTA decoy set, although it does not take into account side chain orientation-dependence explicitly. The DOOP energy function for protein structure prediction, the underlying database of protein structures with hypothetical ligand-receptor systems and their decoys are freely available at http://agknapp.chemie.fu-berlin.de/doop/.

Computing pK(A) values of hexa-aqua transition metal complexes.

Aqueous pKA values for 15 hexa-aqua transition metal complexes were computed using a combination of quantum chemical and electrostatic methods. Two different structure models were considered optimizing the isolated complexes in vacuum or in presence of explicit solvent using a QM/MM approach. They yield very good agreement with experimentally measured pKA values with an overall root mean square deviation of about 1 pH unit, excluding a single but different outlier for each of the two structure models. These outliers are hexa-aqua Cr(III) for the vacuum and hexa-aqua Mn(III) for the QM/MM structure model. Reasons leading to the deviations of the outlier complexes are partially explained. Compared to previous approaches from the same lab the precision of the method was systematically improved as discussed in this study. The refined methods to obtain the appropriate geometries of the complexes, developed in this work, may allow also the computation of accurate pKA values for multicore transition metal complexes in different oxidation states.

pK(A) in proteins solving the Poisson-Boltzmann equation with finite elements.

Knowledge on pK(A) values is an eminent factor to understand the function of proteins in living systems. We present a novel approach demonstrating that the finite element (FE) method of solving the linearized Poisson-Boltzmann equation (lPBE) can successfully be used to compute pK(A) values in proteins with high accuracy as a possible replacement to finite difference (FD) method. For this purpose, we implemented the software molecular Finite Element Solver (mFES) in the framework of the Karlsberg+ program to compute pK(A) values. This work focuses on a comparison between pK(A) computations obtained with the well-established FD method and with the new developed FE method mFES, solving the lPBE using protein crystal structures without conformational changes. Accurate and coarse model systems are set up with mFES using a similar number of unknowns compared with the FD method. Our FE method delivers results for computations of pK(A) values and interaction energies of titratable groups, which are comparable in accuracy. We introduce different thermodynamic cycles to evaluate pK(A) values and we show for the FE method how different parameters influence the accuracy of computed pK(A) values.

ProPairs: A Data Set for Protein-Protein Docking.

ProPairs is a data set of crystal structures of protein complexes defined as biological assemblies in the protein data bank (PDB), which are classified as legitimate protein-protein docking complexes by also identifying the corresponding unbound protein structures in the PDB. The underlying program selecting suitable protein complexes, also called ProPairs, is an automated method to extract structures of legitimate protein docking complexes and their unbound partner proteins from the PDB which fulfill specific criteria. In this way a total of 5,642 protein complexes have been identified with 11,600 different decompositions in unbound protein pairs yielding legitimate protein docking partners. After removing sequence redundancy (requiring a sequence identity of the residues in the interface of less than 40%), 2,070 different legitimate protein docking complexes remain. For 810 of these protein docking complexes, both docking partners possess corresponding unbound structures in the PDB. From the 2,070 nonredundant protein docking complexes there are 417 which possess a cofactor at the interface. From the 176 protein docking complexes of the Protein-Protein Docking Benchmark 4.0 (DB4.0) data set, 13 differ from the ProPairs data set. Twelve of them differ with respect to the composition of the unbound structures but are contained in the large redundant ProPairs data set. One protein docking complex of the DB4.0 data set is not contained in ProPairs since the biological assembly specified in the PDB is wrong (PDB id 1d6r ). For one protein complex (PDB id 1bgx ) the DB4.0 data set uses a fabricated unbound structure. For public use interactive online access is provided to the ProPairs data set of nonredundant protein docking complexes along with the source code of the underlying method [ http://propairs.github.io].

Proton transfer in the K-channel analog of B-type Cytochrome c oxidase from Thermus thermophilus.

A key enzyme in aerobic metabolism is cytochrome c oxidase (CcO), which catalyzes the reduction of molecular oxygen to water in the mitochondrial and bacterial membranes. Substrate electrons and protons are taken up from different sides of the membrane and protons are pumped across the membrane, thereby generating an electrochemical gradient. The well-studied A-type CcO uses two different entry channels for protons: the D-channel for all pumped and two consumed protons, and the K-channel for the other two consumed protons. In contrast, the B-type CcO uses only a single proton input channel for all consumed and pumped protons. It has the same location as the A-type K-channel (and thus is named the K-channel analog) without sharing any significant sequence homology. In this study, we performed molecular-dynamics simulations and electrostatic calculations to characterize the K-channel analog in terms of its energetic requirements and functionalities. The function of Glu-15B as a proton sink at the channel entrance is demonstrated by its rotational movement out of the channel when it is deprotonated and by its high pKA value when it points inside the channel. Tyr-244 in the middle of the channel is identified as the valve that ensures unidirectional proton transfer, as it moves inside the hydrogen-bond gap of the K-channel analog only while being deprotonated. The electrostatic energy landscape was calculated for all proton-transfer steps in the K-channel analog, which functions via proton-hole transfer. Overall, the K-channel analog has a very stable geometry without large energy barriers.

Database of protein complexes with multivalent binding ability: Bival-Bind.

Phenomena of multivalent binding of ligands with receptors are ubiquitous in biology and of growing interest in material sciences. Multivalency can enhance binding affinity dramatically. To understand the mechanism of multivalent binding in more detail model systems of bi- and multivalent receptors are needed, but are difficult to find. Furthermore it is useful to know about multivalent receptors, which can serve as targets to design multivalent drugs. The present contribution tries to close this gap. The Bival-Bind database (http://agknapp.chemie.fu-berlin.de/bivalbind) provides a relatively complete list - 2073 protein complexes with less than 90% sequence identity - out of the protein database, which can serve as bi- or multivalent receptors. Steric clashes of molecular spacers - necessary to connect the monomeric ligand units - with the receptor surface can diminish binding affinity dramatically and, thus, abolish the expected enhancement of binding affinity due to the multivalency. The potential multivalent receptors in the Bival-Bind database are characterized with respect to the receptor surface topography. A height profile between the receptor binding pockets is provided, which is an important information to estimate the influence of unfavorable spacer receptor interaction.

Protein secondary structure classification revisited: processing DSSP information with PSSC.

A first step toward three-dimensional protein structure description is the characterization of secondary structure. The most widely used program for secondary structure assignment remains DSSP, introduced in 1983, with currently more than 400 citations per year. DSSP output is in a one-letter representation, where much of the information on DSSP's internal description is lost. Recently it became evident that DSSP overlooks most π-helical structures, which are more prevalent and important than anticipated before. We introduce an alternative concept, representing the internal structure characterization of DSSP as an eight-character string that is human-interpretable and easy to parse by software. We demonstrate how our protein secondary structure characterization (PSSC) code allows for inspection of complicated structural features. It recognizes ten times more π-helical residues than does the standard DSSP. The plausibility of introduced changes in interpreting DSSP information is demonstrated by better clustering of secondary structures in (φ, ψ) dihedral angle space. With a sliding sequence window (SSW), helical assignments with PSSC remain invariant compared with an assignment based on the complete structure. In contrast, assignment with DSSP can be changed by residues in the neighborhood that are in fact not interacting with the residue under consideration. We demonstrate how one can easily define new secondary structure classification schemes with PSSC and perform the classifications. Our approach works without changing the DSSP source code and allows for more detailed protein characterization.

Charge transport in the ClC-type chloride-proton anti-porter from Escherichia coli.

The first chloride transporter identified in the superfamily of ClC chloride channels was from Escherichia coli (EClC) (Accardi, A., and Miller, C. (2004) Nature 427, 803-807). Pathways, energetics, and mechanism of proton and chloride translocation and their coupling are up to now unclear. To bridge the hydrophobic gap of proton transport, we modeled four stable buried waters into both subunits of the WT EClC structure. Together they form a "water wire" connecting Glu-203 with the chloride at the central site, which in turn connects to Glu-148, the hypothetical proton exit site. Assuming the transient production of hydrochloride in the central chloride binding site of EClC, the water wire could establish a transmembrane proton transport pathway starting from Glu-203 all the way downstream onto Glu-148. We demonstrated by electrostatic and quantum chemical computations that protonation of the central chloride is energetically feasible. We characterized all chloride occupancies and protonation states possibly relevant for the proton-chloride transport cycle in EClC and constructed a working model. Accordingly, EClC evolves through states involving up to two excess protons and between one and three chlorides, which was required to fulfill the experimentally observed 2:1 stoichiometry. We show that the Y445F and E203H mutants of EClC can operate similarly, thus explaining why they exhibit almost WT activity levels. The proposed mechanism of coupled chloride-proton transport in EClC is consistent with available experimental data and allows predictions on the importance of specific amino acids, which may be probed by mutation experiments.

Multivalent binding of formin-binding protein 21 (FBP21)-tandem-WW domains fosters protein recognition in the pre-spliceosome.

The high abundance of repetitive but nonidentical proline-rich sequences in spliceosomal proteins raises the question of how these known interaction motifs recruit their interacting protein domains. Whereas complex formation of these adaptors with individual motifs has been studied in great detail, little is known about the binding mode of domains arranged in tandem repeats and long proline-rich sequences including multiple motifs. Here we studied the interaction of the two adjacent WW domains of spliceosomal protein FBP21 with several ligands of different lengths and composition to elucidate the hallmarks of multivalent binding for this class of recognition domains. First, we show that many of the proteins that define the cellular proteome interacting with FBP21-WW1-WW2 contain multiple proline-rich motifs. Among these is the newly identified binding partner SF3B4. Fluorescence resonance energy transfer (FRET) analysis reveals the tandem-WW domains of FBP21 to interact with splicing factor 3B4 (SF3B4) in nuclear speckles where splicing takes place. Isothermal titration calorimetry and NMR shows that the tandem arrangement of WW domains and the multivalency of the proline-rich ligands both contribute to affinity enhancement. However, ligand exchange remains fast compared with the NMR time scale. Surprisingly, a N-terminal spin label attached to a bivalent ligand induces NMR line broadening of signals corresponding to both WW domains of the FBP21-WW1-WW2 protein. This suggests that distinct orientations of the ligand contribute to a delocalized and semispecific binding mode that should facilitate search processes within the spliceosome.

Oxygen-evolving Mn cluster in photosystem II: the protonation pattern and oxidation state in the high-resolution crystal structure.

Extensive quantum chemical DFT calculations were performed on the high-resolution (1.9 Å) crystal structure of photosystem II in order to determine the protonation pattern and the oxidation states of the oxygen-evolving Mn cluster. First, our data suggest that the experimental structure is not in the S(1)-state. Second, a rather complete set of possible protonation patterns is studied, resulting in very few alternative protonation patterns whose relevance is discussed. Finally, we show that the experimental structure is a mixture of states containing highly reduced forms, with the largest contribution (almost 60%) from the S(-3)-state, Mn(II,II,III,III).