Structure and properties of a ferrocenylpyrimidine-bromanilic acid hydrogen-bonded supramolecular complex

A hydrogen-bonded assembly composed of ferrocenylpyrimidine (FcPM) and bromanilic acid (BA), represented as [FcPM](BA)(acetone)_0.5, was prepared and crystallographically characterized. The asymmetric unit of the crystal contained two crystallographically independent molecules of FcPM and BA, which were alternately connected to form one-dimensional zigzag chains via OH?N hydrogen bonds. The BA molecules were stacked to form one-dimensional columns. No charge transfer was observed between FcPM and BA. Acetone molecules, which were located in channels, were desorbed at 433 K.     

Water-gated mechanism of proton translocation by cytochrome c oxidase

Cytochrome c oxidase is essential for aerobic life as a membrane-bound energy transducer. O₂ reduction at the haem a₃-Cu_B centre consumes electrons transferred via haem a from cytochrome c outside the membrane. Protons are taken up from the inside, both to form water and to be pumped across the membrane (M.K.F. Wikström, Nature 266 (1977) 271 [1]; M. Wikström, K. Krab, M. Saraste, Cytochrome Oxidase, A Synthesis, Academic Press, London, 1981 [2]). The resulting electrochemical proton gradient drives ATP synthesis (P. Mitchell, Chemiosmotic Coupling in Oxidative and Photosynthetic Phosphorylation, Glynn Research, Bodmin, UK, 1966 [3]). Here we present a molecular mechanism for proton pumping coupled to oxygen reduction that is based on the unique properties of water in hydrophobic cavities. An array of water molecules conducts protons from a conserved glutamic acid, either to the Δ-propionate of haem a₃ (pumping), or to haem a₃-Cu_B (water formation). Switching between these pathways is controlled by the redox-state-dependent electric field between haem a and haem a₃-Cu_B, which determines the water-dipole orientation, and therefore the proton transfer direction. Proton transfer via the propionate provides a gate to O₂ reduction. This pumping mechanism explains the unique arrangement of the metal cofactors in the structure. It is consistent with the large body of biochemical data, and is shown to be plausible by molecular dynamics simulations.     

The kinetics of a weakly electron-coupled proton transfer in azurin

The thermodynamics and kinetics of the weakly electron-coupled proton transfer of Pseudomonas aeruginosa azurin have been studied and quantitatively determined using fast scan cyclic voltammetry (CV), in which the protein is adsorbed on two types of electrode (pyrolytic graphite ‘edge’ (PGE) or 1-decanethiol modified gold). Electron transfer is coupled to a slow protonation of His35, which is not a ligand to the copper ion but is located approximately 8 Å away. Protonation of His35 produces small changes in the reduction potential of the copper site, which are time-resolved within the scan rate range 0.01-100 V s⁻¹.     

Energy diagrams and mechanism for proton pumping in cytochrome c oxidase

The powerful technique of energy diagrams has been used to analyze the mechanism for proton pumping in cytochrome c oxidase. Energy levels and barriers are derived starting out from recent kinetic experiments for the O to E transition, and are then refined using general criteria and a few additional experimental facts. Both allowed and non-allowed pathways are obtained in this way. A useful requirement is that the forward and backward rate should approach each other for the full membrane gradient. A key finding is that an electron on heme a (or the binuclear center) must have a significant lowering effect on the barrier for proton uptake, in order to prevent backflow from the pump-site to the N-side. While there is no structural gating in the present mechanism, there is thus an electronic gating provided by the electron on heme a. A quantitative analysis of the energy levels in the diagrams, leads to Prop-A of heme a₃ as the most likely position for the pump-site, and the Glu278 region as the place for the transition state for proton uptake. Variations of key redox potentials and pK_a values during the pumping process are derived for comparison to experiments.     

Exploring the terminal region of the proton pathway in the bacterial nitric oxide reductase

The c-type nitric oxide reductase (cNOR) from Paracoccus (P.) denitrificans is an integral membrane protein that catalyzes NO reduction; 2NO + 2e⁻ + 2H⁺ → N₂O + H₂O. It is also capable of catalyzing the reduction of oxygen to water, albeit more slowly than NO reduction. cNORs are divergent members of the heme-copper oxidase superfamily (HCuOs) which reduce NO, do not pump protons, and the reaction they catalyse is non-electrogenic. All known cNORs have been shown to have five conserved glutamates (E) in the catalytic subunit, by P. denitrificans numbering, the E122, E125, E198, E202 and E267. The E122 and E125 are presumed to face the periplasm and the E198, E202 and E267 are located in the interior of the membrane, close to the catalytic site. We recently showed that the E122 and E125 define the entry point of the proton pathway leading from the periplasm into the active site [U. Flock, F.H. Thorndycroft, A.D. Matorin, D.J. Richardson, N.J. Watmough, P. Ädelroth, J. Biol. Chem. 283 (2008) 3839-3845]. Here we present results from the reaction between fully reduced NOR and oxygen on the alanine variants of the E198, E202 and E267. The initial binding of O₂ to the active site was unaffected by these mutations. In contrast, proton uptake to the bound O₂ was significantly inhibited in both the E198A and E267A variants, whilst the E202A NOR behaved essentially as wildtype. We propose that the E198 and E267 are involved in terminating the proton pathway in the region close to the active site in NOR.     

Glycocardiolipin modulates the surface interaction of the proton pumped by bacteriorhodopsin in purple membrane preparations

Glycocardiolipin is an archaeal analogue of mitochondrial cardiolipin, having an extraordinary affinity for bacteriorhodopsin, the photoactivated proton pump in the purple membrane of Halobacterium salinarum. Here purple membranes have been isolated by osmotic shock from either cells or envelopes of Hbt. salinarum. We show that purple membranes isolated from envelopes have a lower content of glycocardiolipin than standard purple membranes isolated from cells. The properties of bacteriorhodopsin in the two different purple membrane preparations are compared; although some differences in the absorption spectrum and the kinetic of the dark adaptation process are present, the reduction of native membrane glycocardiolipin content does not significantly affect the photocycle (M-intermediate rise and decay) as well as proton pumping of bacteriorhodopsin. However, interaction of the pumped proton with the membrane surface and its equilibration with the aqueous bulk phase are altered.     

The influence of hydrogen bonds on electron transfer rate in photosynthetic RCs

Hydrogen bonds formed between photosynthetic reaction centers (RCs) and their cofactors were shown to affect the efficacy of electron transfer. The mechanism of such influence is determined by sensitivity of hydrogen bonds to electron density rearrangements, which alter hydrogen bonds potential energy surface. Quantum chemistry calculations were carried out on a system consisting of a primary quinone Q_A, non-heme Fe²⁺ ion and neighboring residues_. The primary quinone forms two hydrogen bonds with its environment, one of which was shown to be highly sensitive to the Q_A state. In the case of the reduced primary quinone two stable hydrogen bond proton positions were shown to exist on [Q_A-His^M219] hydrogen bond line, while there is only one stable proton position in the case of the oxidized primary quinone. Taking into account this fact and also the ability of proton to transfer between potential energy wells along a hydrogen bond, theoretical study of temperature dependence of hydrogen bond polarization was carried out. Current theory was successfully applied to interpret dark P⁺/Q_A⁻ recombination rate temperature dependence.     

Bacteriorhodopsin: a high-resolution structural view of vectorial proton transport

Recent 3-D structures of several intermediates in the photocycle of bacteriorhodopsin (bR) provide a detailed structural picture of this molecular proton pump in action. In this review, we describe the sequence of conformational changes of bR following the photoisomerization of its all-trans retinal chromophore, which is covalently bound via a protonated Schiff base to Lys216 in helix G, to a 13-cis configuration. The initial changes are localized near the protein's active site and a key water molecule is disordered. This water molecule serves as a keystone for the ground state of bR since, within the framework of the complex counter ion, it is important both for stabilizing the structure of the extracellular half of the protein, and for maintaining the high pK_a of the Schiff base (the primary proton donor) and the low pK_a of Asp85 (the primary proton acceptor). Subsequent structural rearrangements propagate out from the active site towards the extracellular half of the protein, with a local flex of helix C exaggerating an early movement of Asp85 towards the Schiff base, thereby facilitating proton transfer between these two groups. Other coupled rearrangements indicate the mechanism of proton release to the extracellular medium. On the cytoplasmic half of the protein, a local unwinding of helix G near the backbone of Lys216 provides sites for water molecules to order and define a pathway for the reprotonation of the Schiff base from Asp96 later in the photocycle. A steric clash of the photoisomerized retinal with Trp182 in helix F drives an outward tilt of the cytoplasmic half of this helix, opening the proton transport channel and enabling a proton to be taken up from the cytoplasm. Although bR is the first integral membrane protein to have its catalytic mechanism structurally characterized in detail, several key results were anticipated in advance of the structural model and the general framework for vectorial proton transport has, by and large, been preserved.     

Mechanism of proton transfer through the KC proton pathway in the Vibrio cholerae cbb3 terminal oxidase

The heme?copper oxidases (HCuOs) are terminal components of the respiratory chain, catalyzing oxygen reduction coupled to the generation of a proton motive force. The C-family HCuOs, found in many pathogenic bacteria under low oxygen tension, utilize a single proton uptake pathway to deliver protons both for O₂ reduction and for proton pumping. This pathway, called the K^C-pathway, starts at Glu-49^P in the accessory subunit CcoP, and connects into the catalytic subunit CcoN via the polar residues Tyr-(Y)-227, Asn (N)-293, Ser (S)-244, Tyr (Y)-321 and internal water molecules, and continues to the active site. However, although the residues are known to be functionally important, little is known about the mechanism and dynamics of proton transfer in the K^C-pathway. Here, we studied variants of Y227, N293 and Y321. Our results show that in the N293L variant, proton-coupled electron transfer is slowed during single-turnover oxygen reduction, and moreover it shows a pH dependence that is not observed in wildtype. This suggests that there is a shift in the pK_a of an internal proton donor into an experimentally accessible range, from >10 in wildtype to ~8.8 in N293L. Furthermore, we show that there are distinct roles for the conserved Y321 and Y227. In Y321F, proton uptake from bulk solution is greatly impaired, whereas Y227F shows wildtype-like rates and retains ~50% turnover activity. These tyrosines have evolutionary counterparts in the K-pathway of B-family HCuOs, but they do not have the same roles, indicating diversity in the proton transfer dynamics in the HCuO superfamily.     

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. Proteins 2016; 84:639–654. © 2016 Wiley Periodicals, Inc.     

Dynamics of the glutamic acid 242 side chain in cytochrome c oxidase

In many cytochrome c oxidases glutamic acid 242 is required for proton transfer to the binuclear heme a₃/Cu_B site, and for proton pumping. When present, the side chain of Glu-242 is orientated “down” towards the proton-transferring D-pathway in all available crystal structures. A nonpolar cavity “above” Glu-242 is empty in these structures. Yet, proton transfer from Glu-242 to the binuclear site, and for proton-pumping, is well established, and the cavity has been proposed to at least transiently contain water molecules that would mediate proton transfer. Such proton transfer has been proposed to require isomerisation of the Glu-242 side chain into an “up” position pointing towards the cavity. Here, we have explored the molecular dynamics of the protonated Glu-242 side chain. We find that the “up” position is preferred energetically when the cavity contains four water molecules, but the “down” position is favoured with less water. We conclude that the cavity might be deficient in water in the crystal structures, possibly reflecting the “resting” state of the enzyme, and that the “up/down” equilibrium of Glu-242 may be coupled to the presence of active-site water molecules produced by O₂ reduction.     

The mechanism of proton exclusion in aquaporin channels

The mechanism of proton exclusion in aquaporin channels is elucidated through free energy calculations of the pathway of proton transport. The second generation multistate empirical valence bond (MS-EVB2) model was applied to simulate the interaction of an excess proton with the channel environment. Jarzynski's equality was employed for rapid convergence of the free energy profile. A barrier sufficiently high to block proton transport is located near the channel center at the NPA motif-a site involved in bi-orientational ordering of the embedded water-wire in absence of the excess proton. A second and lower barrier is observed at the selectivity filter near the periplasmic outlet where the channel is narrowest. This secondary barrier may be essential in filtering other large solutes and cations.     

The mechanism of proton transfer between adjacent sites on the molecular surface

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.     

Self-association of glutamic acid-rich fusion peptide analogs of influenza hemagglutinin in the membrane-mimic environments: Effects of positional difference of glutamic acids on side chain ionization constant and intra- and inter-peptide interactions deduced from NMR and gel electrophoresis measurements

Two glutamic acid-rich fusion peptide analogs of influenza hemagglutinin were synthesized to study the organization of the charged peptides in the membranous media. Fluorescence and gel electrophoresis experiments suggested a loose association between the monomers in the vesicles. A model was built which showed that a positional difference of 3, 7 and 4, 8 results in the exposure of Glu3 and Glu7 side chains to the apolar lipidic core. Supportive results include: first, pK_a values of two pH units higher than reference value in aqueous medium for Glu3 and Glu7 CγH, whereas the deviation of pK_a from the reference value for Glu4 and Glu8 CγH is substantially smaller; second, Hill coefficients of titration shift of these protons indicate anti-cooperativity for Glu3 and Glu7 side chain protons but less so for Glu4 and Glu8, implying a strong electrostatic interaction between Glu3 and Glu7 possibly resulting from their localization in an apolar environment; third, positive and larger titration shift for NH of Glu3 is observed compared to that of Glu4, suggesting stronger hydrogen bond between the NH and the carboxylic group of Glu3 than that of Glu4, consistent with higher degree of exposure to hydrophobic medium for the side chain of Glu3.     

Computational studies of proton transport through the M2 channel

The M2 ion channel is an essential component of the influenza A virus. This low-pH gated channel has a high selectivity for protons. Evidence from various experimental data has indicated that the essential structure responsible for the channel is a parallel homo-tetrameric alpha-helix bundle having a left-handed twist with each helix tilted with respect to the membrane normal. A backbone structure has been determined by solid state nuclear magnetic resonance (NMR). Though detailed structures for the side chains are not available yet, evidence has indicated that His37 and Trp41 in the alpha-helix are implicated in the local molecular structure responsible for the selectivity and channel gate. More definitive conformations for the two residues were recently suggested based on the known backbone structure and recently obtained NMR data. While two competitive proton-conductance mechanisms have been proposed, the actual proton-conductance mechanism remains an unsolved problem. Computer simulations of an excess proton in the channel and computational studies of the His37/Trp41 conformations have provided insights into these structural and mechanism issues.     

High resolution crystal structure of Paracoccus denitrificans cytochrome c oxidase: New insights into the active site and the proton transfer pathways

The structure of the two-subunit cytochrome c oxidase from Paracoccus denitrificans has been refined using X-ray cryodata to 2.25 Å resolution in order to gain further insights into its mechanism of action. The refined structural model shows a number of new features including many additional solvent and detergent molecules. The electron density bridging the heme a₃ iron and Cu_B of the active site is fitted best by a peroxo-group or a chloride ion. Two waters or OH⁻ groups do not fit, one water (or OH⁻) does not provide sufficient electron density. The analysis of crystals of cytochrome c oxidase isolated in the presence of bromide instead of chloride appears to exclude chloride as the bridging ligand. In the D-pathway a hydrogen bonded chain of six water molecules connects Asn131 and Glu278, but the access for protons to this water chain is blocked by Asn113, Asn131 and Asn199. The K-pathway contains two firmly bound water molecules, an additional water chain seems to form its entrance. Above the hemes a cluster of 13 water molecules is observed which potentially form multiple exit pathways for pumped protons. The hydrogen bond pattern excludes that the Cu_B ligand His326 is present in the imidazolate form.     

Role of protein motions on proton transfer pathways in human carbonic anhydrase II

We report here a theoretical study on the formation of long-range proton transfer pathways in proteins due to side chain conformational fluctuations of amino acid residues and reorganization of interior hydration positions. The proton transfer pathways in such systems may be modeled as fluctuating hydrogen-bonded networks with both short- and long-lived connections between the networked nodes, the latter being formed by polar protein atoms and water molecules. It is known that these fluctuations may extend over several decades of time ranging from a few femtoseconds to a few milliseconds. We have shown in this article how the use of a variety of theoretical methods may be utilized to detect a generic set of pathways and assess the feasibility of forming one or more transient connections. We demonstrate the application of these methods to the enzyme human carbonic anhydrase II and its mutants. Our results reveal several alternative pathways in addition to the one mediated by His-64. We also probe at length the mechanism of key conformational fluctuations contributing to the formation of the detected pathways.     

How inter-subunit contacts in the membrane domain of complex I affect proton transfer energetics

The respiratory complex I is a redox-driven proton pump that employs the free energy released from quinone reduction to pump protons across its complete ca. 200?Å wide membrane domain. Despite recently resolved structures and molecular simulations, the exact mechanism for the proton transport process remains unclear. Here we combine large-scale molecular simulations with quantum chemical density functional theory (DFT) models to study how contacts between neighboring antiporter-like subunits in the membrane domain of complex I affect the proton transfer energetics. Our combined results suggest that opening of conserved Lys/Glu ion pairs within each antiporter-like subunit modulates the barrier for the lateral proton transfer reactions. Our work provides a mechanistic suggestion for key coupling effects in the long-range force propagation process of complex I.     

The electrochemical proton potential and the proton/electron ratio of the electron transport with fumarate in Wolinella succinogenes

The electrochemical proton potential across the cytoplasmic membrane ( ) as well as the H⁺ / e ratio, which were brought about by the electron transport of Wolinella succinogenes, was measured with the aim of understanding the mechanism of electron-transport-coupled phosphorylation in this anaerobic bacterium. (1) Inverted vesicles derived from the bacterial membrane were found to take up protons from the external medium on initiation of fumarate reduction by H₂. Proton uptake was dependent on the presence of K⁺ within the vesicles, was enhanced by the presence of valinomycin and DCCD and high internal buffer concentration, and was abolished by protonophores. The maximum H⁺ / e ratio slightly exceeded 1. (2) The vesicles accumulated thiocyanate in the steady state of fumarate reduction by H₂. The concentration ratio (internal / external) was close to 1000 at an external thiocyanate concentration below 10 μM. Under the same conditions the uptake of methylamine was negligible. Thiocyanate uptake was abolished by the presence of a protonophore. (3) Cells of W. succinogenes accumulated tetraphenylphosphonium cation (TPP) in the steady state of fumarate reduction with H₂ or formate. Under the same conditions the uptake of benzoic acid was negligible. From the amount of TPP taken up by the bacteria, the free internal concentration of TPP was evaluated according to the procedure of Zaritsky et al. (Zaritsky, A., Kihara, M. and MacNab, R.M. (1981) J. Membrane Biol. 63, 215–231). The concentration ratio (internal / external) was 700 in the absence and close to 1 in the presence of a protonophore or in the absence of external Na⁺. (4) The experimental results are consistent with the view that the energy transduction from electron transport to phosphorylation is done by means of the across the bacterial membrane.