Electron Transfer in Proteins

Electron-transfer (ET) reactions are key steps in a diverse array of biological transformations ranging from photosynthesis to aerobic respiration. A powerful theoretical formalism has been developed that describes ET rates in terms of two parameters: the nuclear reorganization [lambda] energy (1) and the electronic-coupling strength (HAB). Studies of ET reactions in ruthenium-modified proteins have probed [lambda] and HAB in several metalloproteins (cytochrome c, myoglobin, azurin). This work has shown that protein reorganization energies are sensitive to the medium surrounding the redox sites and that an aqueous environment, in particular, leads to large reorganization energies. Analyses of electronic-coupling strengths suggest that the efficiency of long-range ET depends on the protein secondary structure: [beta]sheets appear to mediate coupling more efficiently than [alpha]-helical structures, and hydrogen bonds play a critical role in both

Different types of biological proton transfer reactions studied by quantum chemical methods

AbstractDifferent types of proton transfer occurring in biological systems are described with examples mainly from ribonucleotide reductase (RNR) and cytochrome c oxidase (CcO). Focus is put on situations where electron and proton transfer are rather strongly coupled. In the long range radical transfer in RNR, it is shown that the presence of hydrogen atom transfer (HAT) is the most logical explanation for the experimental observations. In another example from RNR, it is shown that a transition state for concerted motion of both proton and electron can be found even if the donors are separated by a quite long distance. In CcO, the essential proton transfer for the OO bond cleavage, and the most recent modelings of proton translocation are described, indicating a few remaining major problems

Residue coevolution: modeling and interpretation

Coevolution between amino acid residues and its context-dependence are important for exploring protein structure and function, and critical for understanding protein structural and functional evolution. Coevolution has long been ignored because of its complexity and the lack of computing power. In the research presented here, I developed an efficient coevolution analysis methodology based on likelihood comparisons of statistical models. Likelihood ratios and Bayes factors, calculated using the Markov chain Monte Carlo algorithm, were employed as the statistics. Two types of models, 2-state and 3-state, were developed to allow for the context-dependence of coevolution. Computer programs implementing this methodology were coded in C/C++ and were run on the Beowulf clusters of our laboratory and the super computers of LSU. Using these programs and custom Perl scripts, residue coevolution in cytochrome c oxidase and photolyases/cryptochromes protein superfamily was analyzed. I found that pairwise coevolution between residues is highly dependent on protein tertiary structures and functions. I detected extensive coevolving pairs in all our analyses, and these pairs were primary localized in regions of known structural and/or functional importance. I also found that coevolution is related to evolutionary rate and concentrated in moderately conserved sites. In supporting the importance of functional constraints, I detected a non-negligible coevolutionary signal between complex subunits and stronger coevolution in proteins of functional importance. I also found that the interaction between subunits can serve as a local coevolutionary constraint on one subunit rather than driving coevolution between two subunits. Based on coevolutionary patterns, I suggested that a domain without any previously supposed function actually operates as a folding core in the proteins of photolyase/cryptochrome superfamily. The coevolutionary patterns also provided clues regarding the functional evolution of electron transfer in this superfamily. I also found that coevolving sites with double substitutions along a branch tend to occur only at physically contacting sites, and that salt-bridge stabilization and secondary structure stabilization are important forces of residue coevolution. The methodology and programs developed in this research are powerful tools for coevolutionary analysis, which can provide valuable information for characterization of protein structural/functional domains and exploration of protein structural/functional evolution

Insights into functions of the H channel of cytochrome c oxidase from atomistic molecular dynamics simulations

Proton pumping A-type cytochrome c oxidase (CcO) terminates the respiratory chains of mitochondria and many bacteria. Three possible proton transfer pathways (D, K, and H channels) have been identified based on structural, functional, and mutational data. Whereas the D channel provides the route for all pumped protons in bacterial A-type CcOs, studies of bovine mitochondrial CcO have led to suggestions that its H channel instead provides this route. Here, we have studied H-channel function by performing atomistic molecular dynamics simulations on the entire, as well as core, structure of bovine CcO in a lipid-solvent environment. The majority of residues in the H channel do not undergo large conformational fluctuations. Its upper and middle regions have adequate hydration and H-bonding residues to form potential proton-conducting channels, and Asp51 exhibits conformational fluctuations that have been observed crystallographically. In contrast, throughout the simulations, we do not observe transient water networks that could support proton transfer from the N phase toward heme a via neutral His413, regardless of a labile H bond between Ser382 and the hydroxyethylfarnesyl group of heme a. In fact, the region around His413 only became sufficiently hydrated when His413 was fixed in its protonated imidazolium state, but its calculated pK(a) is too low for this to provide the means to create a proton transfer pathway. Our simulations show that the electric dipole moment of residues around heme a changes with the redox state, hence suggesting that the H channel could play a more general role as a dielectric well.Peer reviewe

Proton Pumping in Cytochrome c Oxidase

Cytochrome c oxidase (CcO) is a large trans-membrane protein, which is the final enzyme in the respiratory electron transport chain in mitochondria or aerobic bacteria. It implements proton pumping through the mitochondrial membrane against the electrochemical gradient, by utilizing the chemical energy released by reducing O2 to water. The active site of the chemical reaction is called the Binuclear Center (BNC) that is made up of heme a3, CuB, a Tyrosine residue and their ligands. The protein is reduced four times by electron from cytochromes c to reduce O2 and to generate four different BNC redox states step by step. In each reduction step a proton is delivered to the BNC and another proton is pumped across the protein to increase the trans-membrane proton gradient. In CcO, the pumped proton is firstly located in the proton loading site (PLS), and then is released out of the protein. In these processes, a high conserved Glutamate residue, plays an essential role on the proton translocation either to the BNC or the PLS. In this thesis, Multi-Conformational Continuum Electrostatics (MCCE) and Molecular Dynamics (MD) are combined to study the proton affinity (pKa) of the high conserved Glutamate residue and the identity of the PLS. This Glutamate residue is located in a hydrophobic cavity in the protein, and the simulations show that the hydration of the cavity is controlled by the protonation state of the propionic acid of heme a3, a group on the proton outlet pathway. The changes in hydration and electrostatic interactions lower the proton affinity by at least 5 kcal/mol. The identity of the residues in the PLS is another open question in CcO research, and various groups above the BNC have been considered as candidates. We designed a new model for the simulation via separating the catalytic cycle into smaller substates and monitoring the charge of all residues in the protein. The results demonstrates the PLS is a cluster rather than a single residue, and the proton affinity of the heme a3 propionic acids primarily determines the number of protons loaded into the PLS

B3LYP Study on Reduction Mechanisms from O2 to H2O at the Catalytic Sites of Fully Reduced and Mixed-Valence Bovine Cytochrome c Oxidases

Reduction mechanisms of oxygen molecule to water molecules in the fully reduced (FR) and mixed-valence (MV) bovine cytochrome c oxidases (CcO) have been systematically examined based on the B3LYP calculations. The catalytic cycle using four electrons and four protons has been also shown consistently. The MV CcO catalyses reduction to produce one water molecule, while the FR CcO catalyses to produce two water molecules. One water molecule is added into vacant space between His240 and His290 in the catalytic site. This water molecule constructs the network of hydrogen bonds of Tyr244, farnesyl ethyl, and Thr316 that is a terminal residue of the K-pathway. It plays crucial roles for the proton transfer to the dioxygen to produce the water molecules in both MV and FR CcOs. Tyr244 functions as a relay of the proton transfer from the K-pathway to the added water molecule, not as donors of a proton and an electron to the dioxygen. The reduction mechanisms of MV and FR CcOs are strictly distinguished. In the FR CcO, the Cu atom at the CuB site maintains the reduced state Cu(I) during the process of formation of first water molecule and plays an electron storage. At the final stage of formation of first water molecule, the Cu(I) atom releases an electron to Fe-O. During the process of formation of second water molecule, the Cu atom maintains the oxidized state Cu(II). In contrast with experimental proposals, the K-pathway functions for formation of first water molecule, while the D-pathway functions for second water molecule. The intermediates, PM, PR, F, and O, obtained in this work are compared with those proposed experimentally

Electric fields control water-gated proton transfer in cytochrome c oxidase

Funding Information: ACKNOWLEDGMENTS. This work was funded by the Knut and Alice Wallenberg Foundation (2019.0251 and 2019.0043 to V.R.I.K.). V.R.I.K. also acknowledges support from the German Research Foundation (DFG) via the Collaborative Research Centre (SFB1078) as Mercator Fellow. Computational resources were provided by Funding Information: the Swedish National Infrastructure for Computing (SNIC 2021/1-40, SNIC 2022/1-29) at the Center of High-Performance Computing (PDC), and by the Leibniz-Rechenzentrum. M.W. was supported by the Institute of Biotechnology, University of Helsinki. Funding Information: This work was funded by the Knut and Alice Wallenberg Foundation (2019.0251 and 2019.0043 to V.R.I.K.). V.R.I.K. also acknowledges support from the German Research Foundation (DFG) via the Collaborative Research Centre (SFB1078) as Mercator Fellow. Computational resources were provided by the Swedish National Infrastructure for Computing (SNIC 2021/1-40, SNIC 2022/1-29) at the Center of High-Performance Computing (PDC), and by the Leibniz-Rechenzentrum. M.W. was supported by the Institute of Biotechnology, University of Helsinki. Publisher Copyright: Copyright © 2022 the Author(s). Published by PNAS.Aerobic life is powered by membrane-bound enzymes that catalyze the transfer of electrons to oxygen and protons across a biological membrane. Cytochrome c oxidase (CcO) functions as a terminal electron acceptor in mitochondrial and bacterial respiratory chains, driving cellular respiration and transducing the free energy from O2 reduction into proton pumping. Here we show that CcO creates orientated electric fields around a nonpolar cavity next to the active site, establishing a molecular switch that directs the protons along distinct pathways. By combining large-scale quantum chemical density functional theory (DFT) calculations with hybrid quantum mechanics/ molecular mechanics (QM/MM) simulations and atomistic molecular dynamics (MD) explorations, we find that reduction of the electron donor, heme a, leads to dissociation of an arginine (Arg438)-heme a3 D-propionate ion-pair. This ion-pair dissociation creates a strong electric field of up to 1 V A21 along a water-mediated proton array leading to a transient proton loading site (PLS) near the active site. Protonation of the PLS triggers the reduction of the active site, which in turn aligns the electric field vectors along a second, "chemical," proton pathway. We find a linear energy relationship of the proton transfer barrier with the electric field strength that explains the effectivity of the gating process. Our mechanism shows distinct similarities to principles also found in other energy-converting enzymes, suggesting that orientated electric fields generally control enzyme catalysis.Peer reviewe

A molecular dynamics study of water chain formation in the proton-conducting K channel of cytochrome c oxidase

AbstractThe formation of water chains in cytochrome c oxidase (CcO) is studied by molecular dynamics (MD). Focus is on water chains in the K channel that can supply a proton to the binuclear center (the heme a3 Fe/CuB region), the site of O2 reduction. By assessing the presence of chains of any length on a short time scale (0.1 ps), a view of the kinds of chains and their persistence is obtained. Chains from the entry of the channel on the inner membrane to Thr359 (Rhodobacter sphaeroides numbering) are often present but are blocked at that point until a rotation of the Thr359 side chain occurs, permitting formation of chains from Thr359 towards the binuclear center. No continuous hydrogen-bonded water chains are found connecting Thr359 and the binuclear center. Instead, waters hydrogen bond from Thr359 to the hydroxyl of the heme a3 farnesyl and then continue to the binuclear center via Tyr288, which has been identified as a source of a proton for O2 reduction. Three hydrogen-bonded waters are found to be present in the binuclear center after a sufficiently long simulation time. One is ligated to the CuB and could be associated with a water (or hydroxyl) identified in the crystal structure as the fourth ligand of CuB. The water hydrogen-bonded to the hydroxyl of Tyr288 is extremely persistent and well positioned to participate in O2 reduction. The third water is located where O2 is often suggested to reside in mechanistic studies of O2 reduction