BBA - Bioenergetics (v.1757, #8)

Et tu, Grotthuss! and other unfinished stories by Samuel Cukierman (876-885).
This review article is divided into three sections. In Section 1, a short biographical note on Freiherr von Grotthuss is followed by a detailed summary of the main findings and ideas present in his 1806 paper. Attempts to place Grotthuss contribution in the context of the science done at his time were also made. In Section 2, the modern version of the Grotthuss mechanism is reviewed. The classical Grotthuss model has been recently questioned and new mechanisms and ideas regarding proton transfer are briefly discussed. The last section discusses the significance of a classical Grotthuss mechanism for proton transfer in water chains inside protein cavities. This has been an interesting new twist in the ongoing history of the Grotthuss mechanism. A summary and discussion of what was learned from probably the simplest currently available experimental models of proton transfer in water wires in semi-synthetic ion channels are critically presented. This review ends discussing some of the questions that need to be addressed in the near future.
Keywords: Proton transfer; Membrane; Protein; Water; Single ion channel; Proton current;

Proton transfer and transport in water, gramicidin and some selected channels and bioenergetic proteins are reviewed. An attempt is made to draw some conclusions about how Nature designs long distance, proton transport functionality. The prevalence of water rather than amino acid hydrogen bonded chains is noted, and the possible benefits of waters as the major component are discussed qualitatively.

Protons @ interfaces: Implications for biological energy conversion by Armen Y. Mulkidjanian; Joachim Heberle; Dmitry A. Cherepanov (913-930).
The review focuses on the anisotropy of proton transfer at the surface of biological membranes. We consider (i) the data from “pulsed” experiments, where light-triggered enzymes capture or eject protons at the membrane surface, (ii) the electrostatic properties of water at charged interfaces, and (iii) the specific structural attributes of proton-translocating enzymes. The pulsed experiments revealed that proton exchange between the membrane surface and the bulk aqueous phase takes as much as about 1 ms, but could be accelerated by added mobile pH-buffers. Since the accelerating capacity of the latter decreased with the increase in their electric charge, it was concluded that the membrane surface is separated from the bulk aqueous phase by a barrier of electrostatic nature. The barrier could arise owing to the water polarization at the negatively charged membrane surface. The barrier height depends linearly on the charge of penetrating ions; for protons, it has been estimated as about 0.12 eV. While the proton exchange between the surface and the bulk aqueous phase is retarded by the interfacial barrier, the proton diffusion along the membrane, between neighboring enzymes, takes only microseconds. The proton spreading over the membrane is facilitated by the hydrogen-bonded networks at the surface. The membrane-buried layers of these networks can eventually serve as a storage/buffer for protons (proton sponges). As the proton equilibration between the surface and the bulk aqueous phase is slower than the lateral proton diffusion between the “sources” and “sinks”, the proton activity at the membrane surface, as sensed by the energy transducing enzymes at steady state, might deviate from that measured in the adjoining water phase. This trait should increase the driving force for ATP synthesis, especially in the case of alkaliphilic bacteria.
Keywords: Grotthus mechanism; ATP synthesis; Proton transfer; Membrane potential; Chemiosmotic coupling; Alkaliphilic bacteria; Surface potential; Nonlocal electrostatics;

The mechanism of proton transfer between adjacent sites on the molecular surface by Menachem Gutman; Esther Nachliel; Ran Friedman (931-941).
The surface of a protein, or a membrane, is spotted with a multitude of proton binding sites, some of which are only few Å apart. When a proton is released from one site, it propagates through the water by a random walk under the bias of the local electrostatic potential determined by the distribution of the charges on the protein. Eventually, the released protons are dispersed in the bulk, but during the first few nanoseconds after the dissociation, the protons can be trapped by encounter with nearby acceptor sites. While the study of this reaction on the surface of a protein suffers from experimental and theoretical difficulties, it can be investigated with simple model compounds like derivatives of fluorescein. In the present study, we evaluate the mechanism of proton transfer reactions that proceed, preferentially, inside the Coulomb cage of the dye molecules. Kinetic analysis of the measured dynamics reveals the role of the dimension of the Coulomb cage on the efficiency of the reaction and how the ordering of the water molecules by the dye affects the kinetic isotope effect.
Keywords: Proton Transfer; Kinetic isotope effect; Molecular dynamics; Laser induced proton pulse; Molecular surface; Flourescein; Intra-molecular proton transfer; Coloumb cage;

A protein structure should provide the information needed to understand its observed properties. Significant progress has been made in developing accurate calculations of acid/base and oxidation/reduction reactions in proteins. Current methods and their strengths and weaknesses are discussed. The distribution and calculated ionization states in a survey of proteins is described, showing that a significant minority of acidic and basic residues are buried in the protein and that most of these remain ionized. The electrochemistry of heme and quinones are considered. Proton transfers in bacteriorhodopsin and coupled electron and proton transfers in photosynthetic reaction centers, 5-coordinate heme binding proteins and cytochrome c oxidase are highlighted as systems where calculations have provided insight into the reaction mechanism.
Keywords: Electrostatic; Electrochemistry; pK a; E m; Bacteriorhodopsin; Simulation; Bioenergetics;

Different 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 O―O bond cleavage, and the most recent modelings of proton translocation are described, indicating a few remaining major problems.
Keywords: Quantum chemistry; Density functional theory; Cytochrome c oxidase; Proton transfer; Proton translocation;

Recent data from studies of enzyme catalyzed hydrogen transfer reactions implicate a new theoretical context in which to understand C–H activation. This is much closer to the Marcus theory of electron transfer, in that environmental factors influence the probability of effective wave function overlap from donor to acceptor atoms. The larger size of hydrogen and the availability of three isotopes (H, D and T) introduce a dimension to the kinetic analysis that is not available for electron transfer. This concerns the role of gating between donor and acceptor atoms, in particular whether the system in question is able to tune distance between reactants to achieve maximal tunneling efficiency. Analysis of enzyme systems is providing increasing evidence of a role for active site residues in optimizing the inter-nuclear distance for nuclear tunneling. The ease with which this optimization can be perturbed, through site-specific mutagenesis or an alteration in reaction conditions, is also readily apparent from an analysis of the changes in the temperature dependence of hydrogen isotope effects.

Reconciliation of apparently contradictory experimental results obtained on the quinol: fumarate reductase (QFR), a dihaem-containing respiratory membrane protein complex from Wolinella succinogenes, was previously obtained by the proposal of the so-called E-pathway hypothesis. According to this hypothesis, transmembrane electron transfer via the haem groups is strictly coupled to co-transfer of protons via a transiently established, novel pathway, proposed to contain the side chain of residue Glu-C180 and the distal haem ring-C propionate as the most prominent components. This hypothesis has recently been supported by both theoretical and experimental results. Multiconformation continuum electrostatics calculations predict Glu-C180 to undergo a combination of proton uptake and conformational change upon haem reduction. Strong experimental support for the proposed role of Glu-C180 in the context of the “E-pathway hypothesis” is provided by the effects of replacing Glu-C180 with Gln or Ile by site-directed mutagenesis, the consequences of these mutations for the viability of the resulting mutants, together with the structural and functional characterisation of the corresponding variant enzymes, and the comparison of redox-induced Fourier-transform infrared (FTIR) difference spectra for the wild type and Glu-C180 → Gln variant. A possible haem propionate involvement has recently been supported by combining 13C-haem propionate labelling with redox-induced FTIR difference spectroscopy.
Keywords: Atomic model; FTIR spectroscopy; Fumarate reductase; Isotope labelling; Membrane protein; Transmembrane electrochemical proton potential; X-ray crystallography;

Charge compensation during the phagocyte respiratory burst by Ricardo Murphy; Thomas E. DeCoursey (996-1011).
The phagocyte NADPH oxidase produces superoxide anion (O2 ·−) by the electrogenic process of moving electrons across the cell membrane. This charge translocation must be compensated to prevent self-inhibition by extreme membrane depolarization. Examination of the mechanisms of charge compensation reveals that these mechanisms perform several other vital functions beyond simply supporting oxidase activity. Voltage-gated proton channels compensate most of the charge translocated by the phagocyte NADPH oxidase in human neutrophils and eosinophils. Quantitative modeling of NADPH oxidase in the plasma membrane supports this conclusion and shows that if any other conductance is present, it must be miniscule. In addition to charge compensation, proton flux from the cytoplasm into the phagosome (a) helps prevent large pH excursions both in the cytoplasm and in the phagosome, (b) minimizes osmotic disturbances, and (c) provides essential substrate protons for the conversion of O2 ·− to H2O2 and then to HOCl. A small contribution by K+ or Cl fluxes may offset the acidity of granule contents to keep the phagosome pH near neutral, facilitating release of bactericidal enzymes. In summary, the mechanisms used by phagocytes for charge compensation during the respiratory burst would still be essential to phagocyte function, even if NADPH oxidase were not electrogenic.
Keywords: Respiratory burst; Phagocyte; Proton channel; NADPH oxidase; Electron current; pH;

The steps in the mechanism of proton transport in bacteriorhodopsin include examples for most kinds of proton transfer reactions that might occur in a transmembrane pump: proton transfer via a bridging water molecule, coupled protonation/deprotonation of two buried groups separated by a considerable distance, long-range proton migration over a hydrogen-bonded aqueous chain, and capture as well as release of protons at the membrane–water interface. The conceptual and technical advantages of this system have allowed close examination of many of these model reactions, some at an atomic level.
Keywords: Bacteriorhodopsin; Retinal; X-ray structure; Proton release; Proton uptake; Hydrogen-bonded chain;

Proton pumping in the bc1 complex: A new gating mechanism that prevents short circuits by Antony R. Crofts; Sangmoon Lhee; Stephanie B. Crofts; Jerry Cheng; Stuart Rose (1019-1034).
The Q-cycle mechanism of the bc1 complex explains how the electron transfer from ubihydroquinone (quinol, QH2) to cytochrome (cyt) c (or c2 in bacteria) is coupled to the pumping of protons across the membrane. The efficiency of proton pumping depends on the effectiveness of the bifurcated reaction at the Qo-site of the complex. This directs the two electrons from QH2 down two different pathways, one to the high potential chain for delivery to an electron acceptor, and the other across the membrane through a chain containing heme bL and bH to the Qi-site, to provide the vectorial charge transfer contributing to the proton gradient. In this review, we discuss problems associated with the turnover of the bc1 complex that center around rates calculated for the normal forward and reverse reactions, and for bypass (or short-circuit) reactions. Based on rate constants given by distances between redox centers in known structures, these appeared to preclude conventional electron transfer mechanisms involving an intermediate semiquinone (SQ) in the Qo-site reaction. However, previous research has strongly suggested that SQ is the reductant for O2 in generation of superoxide at the Qo-site, introducing an apparent paradox. A simple gating mechanism, in which an intermediate SQ mobile in the volume of the Qo-site is a necessary component, can readily account for the observed data through a coulombic interaction that prevents SQ anion from close approach to heme bL when the latter is reduced. This allows rapid and reversible QH2 oxidation, but prevents rapid bypass reactions. The mechanism is quite natural, and is well supported by experiments in which the role of a key residue, Glu-295, which facilitates proton transfer from the site through a rotational displacement, has been tested by mutation.
Keywords: Q-cycle; bc1 complex; Proton circuit; Rhodobacter sphaeroides; Qo-site; Superoxide; –PEWY–span;

Combined DFT and electrostatics study of the proton pumping mechanism in cytochrome c oxidase by Jason Quenneville; Dragan M. Popović; Alexei A. Stuchebrukhov (1035-1046).
Cytochrome c oxidase is a redox-driven proton pump which converts atmospheric oxygen to water and couples the oxygen reduction reaction to the creation of a membrane proton gradient. The structure of the enzyme has been solved; however, the mechanism of proton pumping is still poorly understood. Recent calculations from this group indicate that one of the histidine ligands of enzyme's CuB center, His291, may play the role of the pumping element. In this paper, we report on the results of calculations that combined first principles DFT and continuum electrostatics to evaluate the energetics of the key energy generating step of the model—the transfer of the chemical proton to the binuclear center of the enzyme, where the hydroxyl group is converted to water, and the concerted expulsion of the proton from δ-nitrogen of His291 ligand of CuB center. We show that the energy generated in this step is sufficient to push a proton against an electrochemical membrane gradient of about 200 mV. We have also re-calculated the pK a of His291 for an extended model in which the whole Fe a3–CuB center with their ligands is treated by DFT. Two different DFT functionals (B3LYP and PBE0), and various dielectric models of the protein have been used in an attempt to estimate potential errors of the calculations. Although current methods of calculations do not allow unambiguous predictions of energetics in proteins within few pK a units, as required in this case, the present calculation provides further support for the proposed His291 model of CcO pump and makes a specific prediction that could be targeted in the experimental test.
Keywords: Cytochrome c oxidase; DFT/electrostatic calculation; Proton pumping; Redox-coupled pK a; pK a calculation;

Towards the mechanism of proton pumping by the haem-copper oxidases by Mårten Wikström; Michael I. Verkhovsky (1047-1051).
The haem-copper oxidases comprise a large family of enzymes that is widespread among aerobic organisms. These remarkable membrane-bound proteins catalyse the respiratory reduction of dioxygen to water, and conserve free energy from this reaction by operating as proton pumps. The mechanism of redox-dependent proton translocation has been elusive despite the availability of high resolution crystal structures from several oxidases. Here, we discuss some recent as well as some older results that may shed light on this mechanism. We conclude that proton-pumping is initiated by vectorial proton transfer from a conserved glutamic acid (Glu242 in the bovine enzyme) to a proton acceptor above the haem groups, and that this primary event is mechanistically coupled to electron transfer from haem a to the binuclear haem a 3/CuB centre. Subsequently, Glu242 is reprotonated from the negatively charged side of the membrane. Next this proton is transferred to the binuclear site to complete the chemistry, Glu242 is reprotonated once more, and the “prepumped” proton is ejected on the opposite side of the membrane. The different kinetics of electron-coupled proton transfer in different steps of the catalytic cycle may be related to differences in the driving force due to different E m values of the electron acceptor in the binuclear site.
Keywords: Electron transfer; Proton transfer; Energy transduction; Cell respiration;

Transmembrane proton translocation by cytochrome c oxidase by Gisela Brändén; Robert B. Gennis; Peter Brzezinski (1052-1063).
Respiratory heme-copper oxidases are integral membrane proteins that catalyze the reduction of molecular oxygen to water using electrons donated by either quinol (quinol oxidases) or cytochrome c (cytochrome c oxidases, CcOs). Even though the X-ray crystal structures of several heme-copper oxidases and results from functional studies have provided significant insights into the mechanisms of O2-reduction and, electron and proton transfer, the design of the proton-pumping machinery is not known. Here, we summarize the current knowledge on the identity of the structural elements involved in proton transfer in CcO. Furthermore, we discuss the order and timing of electron-transfer reactions in CcO during O2 reduction and how these reactions might be energetically coupled to proton pumping across the membrane.
Keywords: Cytochrome aa 3; Quinol oxidase; Electron transfer; Proton transfer; Electrochemical proton gradient;