Understanding the cytochrome c oxidase proton pump: thermodynamics of redox linkage.

The cytochrome c oxidase complex (CcO) catalyzes the four-electron reduction of dioxygen to water by using electrons from ferrocytochrome c. Redox free energy released in this highly exergonic process is utilized to drive the translocation of protons across a transmembrane electrochemical gradient. Although numerous chemical models of proton pumping have been developed, few attempts have been made to explain the stepwise transfer of energy in the context of proposed protein conformational changes. A model is described that seeks to clarify the thermodynamics of the proton pumping function of CcO and that illustrates the importance of electron and proton gating to prevent the occurrence of the more exergonic electron leak and proton slip reactions. The redox energy of the CcO-membrane system is formulated in terms of a multidimensional energy surface projected into two dimensions, a nuclear coordinate associated with electron transfer and a nuclear coordinate associated with elements of the proton pump. This model provides an understanding of how a transmembrane electrochemical gradient affects the efficiency of the proton pumping process. Specifically, electron leak and proton slip reactions become kinetically viable as a result of the greater energy barriers that develop for the desired reactions in the presence of a transmembrane potential.     

Estimation of transmembrane pH gradients from phase equilibria of spin-labeled amines.

Spin-labeled secondary amines have been used to measure transmembrane proton gradients in sonicated liposomes. The electron paramagnetic resonance spectra of these probes show changes in the ratio of membrane associated to free aqueous probe as a function of transmembrane pH gradient. As the pH gradient is increased, inside acidic, the amount of membrane associated probe increases. The results are accounted for by a simple thermodynamic theory.     

Thermodynamic stability of water molecules in the bacteriorhodopsin proton channel: a molecular dynamics free energy perturbation study.

The proton transfer activity of the light-driven proton pump, bacteriorhodopsin (bR) in the photochemical cycle might imply internal water molecules. The free energy of inserting water molecules in specific sites along the bR transmembrane channel has been calculated using molecular dynamics simulations based on a microscopic model. The existence of internal hydration is related to the free energy change on transfer of a water molecule from bulk solvent into a specific binding site. Thermodynamic integration and perturbation methods were used to calculate free energies of hydration for each hydrated model from molecular dynamics simulations of the creation of water molecules into specific protein-binding sites. A rigorous statistical mechanical formulation allowing the calculation of the free energy of transfer of water molecules from the bulk to a protein cavity is used to estimate the probabilities of occupancy in the putative bR proton channel. The channel contains a region lined primarily by nonpolar side-chains. Nevertheless, the results indicate that the transfer of four water molecules from bulk water to this apparently hydrophobic region is thermodynamically permitted. The column forms a continuous hydrogen-bonded chain over 12 A between a proton donor, Asp 96, and the retinal Schiff base acceptor. The presence of two water molecules in direct hydrogen-bonding association with the Schiff base is found to be strongly favorable thermodynamically. The implications of these results for the mechanism of proton transfer in bR are discussed.     

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

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

What really prevents proton transport through aquaporin? Charge self-energy versus proton wire proposals

The nature of the control of water/proton selectivity in biological channels is a problem of a fundamental importance. Most studies of this issue have proposed that an interference with the orientational requirements of the so-called proton wire is the source of selectivity. The elucidation of the structures of aquaporins, which have evolved to prevent proton transfer (PT), provided a clear benchmark for exploring the selectivity problem. Previous simulations of this system have not examined, however, the actual issue of PT, but only considered the much simpler task of the transfer of water molecules. Here we take aquaporin as a benchmark and quantify the origin of the water/proton selectivity in this and related systems. This is done by evaluating in a consistent way the free energy profile for transferring a proton along the channel and relating this profile to the relevant PT rate constants. It is found that the water/proton selectivity is controlled by the change in solvation free energy upon moving the charged proton from water to the channel. The reason for the focus on the elegant concept of the proton wire and the related Grotthuss-type mechanism is also considered. It is concluded that these mechanisms are clearly important in cases with flat free energy surfaces (e.g., in bulk water, in gas phase water chains, and in infinitely long channels). However, in cases of biological channels, the actual PT mechanism is much less important than the energetics of transferring the proton charge from water to different regions in the channels.     

Proton-transport mechanisms in cytochrome c oxidase revealed by studies of kinetic isotope effects

Cytochrome c oxidase (CytcO) is a membrane-bound enzyme, which catalyzes the reduction of di-oxygen to water and uses a major part of the free energy released in this reaction to pump protons across the membrane. In the Rhodobacter sphaeroides aa? CytcO all protons that are pumped across the membrane, as well as one half of the protons that are used for O? reduction, are transferred through one specific intraprotein proton pathway, which holds a highly conserved Glu286 residue. Key questions that need to be addressed in order to understand the function of CytcO at a molecular level are related to the timing of proton transfers from Glu286 to a "pump site" and the catalytic site, respectively. Here, we have investigated the temperature dependencies of the H/D kinetic-isotope effects of intramolecular proton-transfer reactions in the wild-type CytcO as well as in two structural CytcO variants, one in which proton uptake from solution is delayed and one in which proton pumping is uncoupled from O? reduction. These processes were studied for two specific reaction steps linked to transmembrane proton pumping, one that involves only proton transfer (peroxy-ferryl, P→F, transition) and one in which the same sequence of proton transfers is also linked to electron transfer to the catalytic site (ferryl-oxidized, F→O, transition). An analysis of these reactions in the framework of theory indicates that that the simpler, P→F reaction is rate-limited by proton transfer from Glu286 to the catalytic site. When the same proton-transfer events are also linked to electron transfer to the catalytic site (F→O), the proton-transfer reactions might well be gated by a protein structural change, which presumably ensures that the proton-pumping stoichiometry is maintained also in the presence of a transmembrane electrochemical gradient. Furthermore, the present study indicates that a careful analysis of the temperature dependence of the isotope effect should help us in gaining mechanistic insights about CytcO.     

Properties of intramolecular proton transfer in carbonic anhydrase III.

We investigated the efficiency of glutamic acid 64 and aspartic acid 64 as proton donors to the zinc-bound hydroxide in a series of site-specific mutants of human carbonic anhydrase III (HCA III). Rate constants for this intramolecular proton transfer, a step in the catalyzed dehydration of bicarbonate, were determined from the proton-transfer-dependent rates of release of H2 18O from the enzyme measured by mass spectrometry. The free energy plots representing these rate constants could be fit by the Marcus rate theory, resulting in an intrinsic barrier for the proton transfer of deltaG0++ = 2.2 +/- 0.5 kcal/mol, and a work function or thermodynamic contribution to the free energy of reaction wr = 10.8 +/- 0.1 kcal/mol. These values are very similar in magnitude to the Marcus parameters describing intramolecular proton transfer from His64 and His67 to the zinc-bound hydroxide in mutants of HCA III. That result and the equivalent efficiency of Glu64 and Asp64 as proton donors in the catalysis by CA III demonstrate a lack of specificity in proton transfer from these sites, which is indirect evidence of a number of proton conduction pathways through different structures of intervening water chains. The dominance of the thermodynamic contribution or work function for all of these proton transfers is consistent with the view that formation and breaking of hydrogen bonds in such water chains is a limiting factor for proton translocation.     

Molecular modeling of the bacterial outer membrane receptor energizer, ExbBD/TonB, based on homology with the flagellar motor, MotAB

The MotA/MotB proteins serve as the motor that drives bacterial flagellar rotation in response to the proton motive force (pmf). They have been shown to comprise a transmembrane proton pathway. The ExbB/ExbD/TonB protein complex serves to energize transport of iron siderophores and vitamin B₁₂ across the outer membrane of the Gram-negative bacterial cell using the pmf. These two protein complexes have the same topology and are homologous. Based on molecular data for the MotA/MotB proteins, we propose simple three-dimensional channel structures for both MotA/MotB and ExbB/ExbD/TonB using modeling methods. Features of the derived channels are discussed, and two possible proton transfer pathways for the ExbBD/TonB system are proposed. These analyses provide a guide for molecular studies aimed at elucidating the mechanism by which chemiosmotic energy can be transferred either between two adjacent membranes to energize outer membrane transport or to the bacterial flagellum to generate torque.     

Molecular modeling of the bacterial outer membrane receptor energizer,ExbBD/TonB,based on homology with the flagellar motor,MotAB

The MotA/MotB proteins serve as the motor that drives bacterial flagellar rotation in response to the proton motive force (pmf). They have been shown to comprise a transmembrane proton pathway. The ExbB/ExbD/TonB protein complex serves to energize transport of iron siderophores and vitamin B12 across the outer membrane of the Gram-negative bacterial cell using the pmf. These two protein complexes have the same topology and are homologous. Based on molecular data for the MotA/MotB proteins, we propose simple three-dimensional channel structures for both MotA/MotB and ExbB/ExbD/TonB using modeling methods. Features of the derived channels are discussed, and two possible proton transfer pathways for the ExbBD/TonB system are proposed. These analyses provide a guide for molecular studies aimed at elucidating the mechanism by which chemiosmotic energy can be transferred either between two adjacent membranes to energize outer membrane transport or to the bacterial flagellum to generate torque.     

Potentials of mean force in acidic proton transfer reactions in constrained geometries

Free energy barriers associated with the transfer of an excess proton in water and related to the potentials of mean force in proton transfer episodes have been computed in a wide range of thermodynamic states, from low-density amorphous ices to high-temperature liquids under the critical point for unconstrained and constrained systems. The latter were represented by set-ups placed inside hydrophobic graphene slabs at the nanometric scale allocating a few water layers, namely one or two in the narrowest case. Water–proton and carbon–proton forces were modelled with a Multi-State Empirical Valence Bond method. As a general trend, a competition between the effects of confinement and temperature is observed on the local hydrogen-bonded structures around the lone proton and, consequently, on the mean force exerted by its environment on the water molecule carrying the proton. Free energy barriers estimated from the computed potentials of mean force tend to rise with the combined effect of increasing temperatures and the packing effect due to a larger extent of hydrophobic confinement. The main reason observed for such enhancement of the free energy barriers was the breaking of the second coordination shell around the lone proton.     

Fixing the Q cycle

Mitchell's key insight that all bioenergetic membranes run on the conversion of redox energy into transmembrane electrical and proton gradients took the form 30 years ago of a working model of the Q cycle of cytochrome bc1, which operates reversibly on coupled electron and proton transfers of quinone at two binding sites on opposite membrane faces. His remarkable model still stands today, but he had no structural information to provide understanding into how dangerous short-circuit redox reactions were avoided. Now, it is clear that the Q cycle must be fixed with a special mechanism that avoids semiquinone-mediated short circuits. Either the redox states of the quinone electron-transfer partners double-gate the semiquinone-intermediate stability, or semiquinone is avoided altogether in concerted double-electron transfer.     

Intervesicular exchange of lipids with weak acid and weak base characteristics: influence of transmembrane pH gradients

Transmembrane pH gradients have previously been shown to induce an asymmetric transmembrane distribution of simple lipids that exhibit weak acid or basic characteristics (Hope, M.J. and Cullis, P.R. (1987) J. Biol. Chem. 262, 4360-4366). In the present study we have examined the influence of proton gradients on the inter-vesicular exchange of stearylamine and oleic acid. We show that vesicles containing stearylamine immediately aggregate with vesicles containing phosphatidylserine and that disaggregation occurs subsequently as stearylamine equilibrates between the two vesicle populations. Despite visible flocculation during the aggregation phase, vesicle integrity is maintained. Stearylamine is the only lipid to exchange, fusion does not occur and vesicles are able to maintain a proton gradient. When stearylamine is sequestered to the inner monolayer in response to a transmembrane pH gradient (inside acidic) aggregation is not observed and diffusion of stearylamine to acceptor vesicles is greatly reduced. The ability of delta pH-dependent lipid asymmetry to modulate lipid exchange is also demonstrated for fatty acids. Oleic acid can be induced to transfer from one population of vesicles to another by maintaining a basic interior pH in the acceptor vesicles. Moreover, it is shown that the same acceptor vesicles are capable of depleting serum albumin of bound fatty acid. These results are discussed with respect to the mechanism and modulation of lipid flow between membranes both in vitro and in vivo.     

Evidence for transmembrane proton transfer in a dihaem-containing membrane protein complex

Membrane protein complexes can support both the generation and utilisation of a transmembrane electrochemical proton potential ('proton-motive force'), either by transmembrane electron transfer coupled to protolytic reactions on opposite sides of the membrane or by transmembrane proton transfer. Here we provide the first evidence that both of these mechanisms are combined in the case of a specific respiratory membrane protein complex, the dihaem-containing quinol:fumarate reductase (QFR) of Wolinella succinogenes, so as to facilitate transmembrane electron transfer by transmembrane proton transfer. We also demonstrate the non-functionality of this novel transmembrane proton transfer pathway ('E-pathway') in a variant QFR where a key glutamate residue has been replaced. The 'E-pathway', discussed on the basis of the 1.78-Angstrom-resolution crystal structure of QFR, can be concluded to be essential also for the viability of pathogenic varepsilon-proteobacteria such as Helicobacter pylori and is possibly relevant to proton transfer in other dihaem-containing membrane proteins, performing very different physiological functions.     

Sodium,protons, and energy coupling in the methanogenic bacteria

In this review, I focus on the bioenergetics of the methanogenic bacteria, with particular attention directed to the roles of transmembrane electrochemical gradients of sodium and proton. In addition, the mechanism of coupling ATP synthesis to methanogenic electron transfer is addressed. Evidence is reviewed which suggests that the methanogens possess great diversity in their bioenergetic machinery. In particular, in some methanogens the primary ion which is translocated coupled to metabolic energy is the proton, while others appear to utilize sodium. In addition, ATP synthesis driven by methanogenic electron transfer is accomplished in some organisms by a chemiosmotic mechanism and is coupled by a more direct mechanism in others. A possible explanation for this diversity (which is consistent with the relatedness of these organisms to each other and to other members of the Archaebacteria as determined by molecular biological techniques) is discussed.     

The influence of a transmembrane pH gradient on protonation probabilities of bacteriorhodopsin: the structural basis of the back-pressure effect

Bacteriorhodopsin pumps protons across a membrane using the energy of light. The proton pumping is inhibited when the transmembrane proton gradient that the protein generates becomes larger than four pH units. This phenomenon is known as the back-pressure effect. Here, we investigate the structural basis of this effect by predicting the influence of a transmembrane pH gradient on the titration behavior of bacteriorhodopsin. For this purpose we introduce a method that accounts for a pH gradient in protonation probability calculations. The method considers that in a transmembrane protein, which is exposed to two different aqueous phases, each titratable residue is accessible for protons from one side of the membrane depending on its hydrogen-bond pattern. This method is applied to several ground-state structures of bacteriorhodopsin, which residues already present complicated titration behaviors in the absence of a proton gradient. Our calculations show that a pH gradient across the membrane influences in a non-trivial manner the protonation probabilities of six titratable residues which are known to participate in the proton transfer: D85, D96, D115, E194, E204, and the Schiff base. The residues connected to one side of the membrane are influenced by the pH on the other side because of their long-range electrostatic interactions within the protein. In particular, D115 senses the pH at the cytoplasmic side of the membrane and transmits this information to D85 and the Schiff base. We propose that the strong electrostatic interactions found between D85, D115, and the Schiff base as well as the interplay of their respective protonation states under the influence of a transmembrane pH gradient are responsible for the back-pressure effect on bacteriorhodopsin.     

Functional proton transfer pathways in the heme–copper oxidase superfamily

Heme–copper oxidases (HCuOs) terminate the respiratory chain in mitochondria and most bacteria. They are transmembrane proteins that catalyse the reduction of oxygen and use the liberated free energy to maintain a proton-motive force across the membrane. The HCuO superfamily has been divided into the oxygen-reducing A-, B- and C-type oxidases as well as the bacterial NO reductases (NOR), catalysing the reduction of NO in the denitrification process. Proton transfer to the catalytic site in the mitochondrial-like A family occurs through two well-defined pathways termed the D- and K-pathways. The B, C, and NOR families differ in the pathways as well as the mechanisms for proton transfer to the active site and across the membrane. Recent structural and functional investigations, focussing on proton transfer in the B, C and NOR families will be discussed in this review. This article is part of a Special Issue entitled: Respiratory Oxidases.     

Functional proton transfer pathways in the heme-copper oxidase superfamily

Heme-copper oxidases (HCuOs) terminate the respiratory chain in mitochondria and most bacteria. They are transmembrane proteins that catalyse the reduction of oxygen and use the liberated free energy to maintain a proton-motive force across the membrane. The HCuO superfamily has been divided into the oxygen-reducing A-, B- and C-type oxidases as well as the bacterial NO reductases (NOR), catalysing the reduction of NO in the denitrification process. Proton transfer to the catalytic site in the mitochondrial-like A family occurs through two well-defined pathways termed the D- and K-pathways. The B, C, and NOR families differ in the pathways as well as the mechanisms for proton transfer to the active site and across the membrane. Recent structural and functional investigations, focussing on proton transfer in the B, C and NOR families will be discussed in this review.     

Characterization of transmembrane movement of glucose and glucose analogs in Streptococcus mutants Ingbritt.

The transmembrane movement of radiolabeled, nonmetabolizable glucose analogs in Streptococcus mutants Ingbritt was studied under conditions of differing transmembrane electrochemical potentials (delta psi) and pH gradients (delta pH). The delta pH and delta psi were determined from the transmembrane equilibration of radiolabeled benzoate and tetraphenylphosphonium ions, respectively. Growth conditions of S. mutants Ingbritt were chosen so that the cells had a low apparent phosphoenolpyruvate (PEP)-dependent glucose:phosphotransferase activity. Cells energized under different conditions produced transmembrane proton potentials ranging from -49 to -103 mV but did not accumulate 6-deoxyglucose intracellularly. An artificial transmembrane proton potential was generated in deenergized cells by creating a delta psi with a valinomycin-induced K+ diffusion potential and a delta pH by rapid acidification of the medium. Artificial transmembrane proton potentials up to -83 mV, although producing proton influx, could not accumulate 6-deoxyglucose in deenergized cells or 2-deoxyglucose or thiomethylgalactoside in deenergized, PEP-depleted cells. The transmembrane diffusion of glucose in PEP-depleted, KF-treated cells did not exhibit saturation kinetics or competitive inhibition by 6-deoxyglucose or 2-deoxyglucose, indicating that diffusion was not facilitated by a membrane carrier. As proton-linked membrane carriers have been shown to facilitate diffusion in the absence of a transmembrane proton potential, the results therefore are not consistent with a proton-linked glucose carrier in S. mutans Ingbritt. This together with the lack of proton-linked transport of the glucose analogs suggests that glucose transmembrane movement in S. mutans Ingbritt is not linked to the transmembrane proton potential.     

Coarse-grained molecular dynamics simulations of the energetics of helix insertion into a lipid bilayer

Experimental and computational studies have indicated that hydrophobicity plays a key role in driving the insertion of transmembrane alpha-helices into lipid bilayers. Molecular dynamics simulations allow exploration of the nature of the interactions of transmembrane alpha-helices with their lipid bilayer environment. In particular, coarse-grained simulations have considerable potential for studying many aspects of membrane proteins, ranging from their self-assembly to the relation between their structure and function. However, there is a need to evaluate the accuracy of coarse-grained estimates of the energetics of transmembrane helix insertion. Here, three levels of complexity of model system have been explored to enable such an evaluation. First, calculated free energies of partitioning of amino acid side chains between water and alkane yielded an excellent correlation with experiment. Second, free energy profiles for transfer of amino acid side chains along the normal to a phosphatidylcholine bilayer were in good agreement with experimental and atomistic simulation studies. Third, estimation of the free energy profile for transfer of an arginine residue, embedded within a hydrophobic alpha-helix, to the center of a lipid bilayer gave a barrier of approximately 15 kT. Hence, there is a substantial barrier to membrane insertion for charged amino acids, but the coarse-grained model still underestimates the corresponding free energy estimate (approximately 29 kT) from atomistic simulations (Dorairaj, S., and Allen, T. W. (2007) Proc. Natl. Acad. Sci. U.S.A. 104, 4943-4948). Coarse-grained simulations were then used to predict the free energy profile for transfer of a simple model transmembrane alpha-helix (WALP23) across a lipid bilayer. The results indicated that a transmembrane orientation was favored by about -70 kT.     

Analysis of bacterial flagellar rotation

Bacterial flagella have rotary motors at their base; embedded in the cytoplasmic membrane and powered by transmembrane ion gradients instead of ATP. Assays have been developed to measure the torque output of individual motors over a wide regime of load, to correlate the energizing proton flux with rotation speed and relate through genetic analysis motor structure to function. These assays promise substantial advances in understanding mechanochemical coupling in these motors. Here, I summarize the present status of our understanding of energy transduction in bacterial flagella and compare this with the case for muscle.