The role of protein-bound water molecules in microbial rhodopsins

Protein-bound internal water molecules are essential features of the structure and function of microbial rhodopsins. Besides structural stabilization, they act as proton conductors and even proton storage sites. Currently, the most understood model system exhibiting such features is bacteriorhodopsin (bR). During the last 20 years, the importance of water molecules for proton transport has been revealed through this protein. It has been shown that water molecules are as essential as amino acids for proton transport and biological function. In this review, we present an overview of the historical development of this research on bR. We furthermore summarize the recently discovered protein-bound water features associated with proton transport. Specifically, we discuss a pentameric water/amino acid arrangement close to the protonated Schiff base as central proton-binding site, a protonated water cluster as proton storage site at the proton-release site, and a transient linear water chain at the proton uptake site. We highlight how protein conformational changes reposition or reorient internal water molecules, thereby guiding proton transport. Last, we compare the water positions in bR with those in other microbial rhodopsins to elucidate how protein-bound water molecules guide the function of microbial rhodopsins. This article is part of a Special Issue entitled: Retinal Proteins — You can teach an old dog new tricks.     

Mutational widening of constrictions in a formate–nitrite/H+ transporter enables aquaporin-like water permeability and proton conductance

The unrelated protein families of the microbial formate–nitrite transporters (FNTs) and aquaporins (AQP) likely adapted the same protein fold through convergent evolution. FNTs facilitate weak acid anion/H⁺ cotransport, whereas AQP water channels strictly exclude charged substrates including protons. The FNT channel–like transduction pathway bears two lipophilic constriction sites that sandwich a highly conserved histidine residue. Because of lacking experiments, the function of these constrictions is unclear, and the protonation status of the central histidine during substrate transport remains a matter of debate. Here, we introduced constriction-widening mutations into the prototypical FNT from Escherichia coli, FocA, and assayed formate/H⁺ transport properties, water/solute permeability, and proton conductance. We found that enlargement of these constrictions concomitantly decreased formate/formic acid transport. In contrast to wildtype FocA, the mutants were unable to make use of a transmembrane proton gradient as a driving force. A construct in which both constrictions were eliminated exhibited water permeability, similar to AQPs, although accompanied by a proton conductance. Our data indicate that the lipophilic constrictions mainly act as barriers to isolate the central histidine from the aqueous bulk preventing protonation via proton wires. These results are supportive of an FNT transport model in which the central histidine is uncharged, and weak acid substrate anion protonation occurs in the vestibule regions of the transporter before passing the constrictions.     

Mechanism of formate–nitrite transporters by dielectric shift of substrate acidity

Bacterial formate–nitrite transporters (FNTs) regulate the metabolic flow of small, weak mono-acids. Recently, the eukaryotic PfFNT was identified as the malaria parasite's lactate transporter and novel drug target. Despite crystal data, central mechanisms of FNT gating and transport remained unclear. Here, we show elucidation of the FNT transport mechanism by single-step substrate protonation involving an invariant lysine in the periplasmic vestibule. Opposing earlier gating hypotheses and electrophysiology reports, quantification of total uptake by radiolabeled substrate indicates a permanently open conformation of the bacterial formate transporter, FocA, irrespective of the pH. Site-directed mutagenesis, heavy water effects, mathematical modeling, and simulations of solvation imply a general, proton motive force-driven FNT transport mechanism: Electrostatic attraction of the acid anion into a hydrophobic vestibule decreases substrate acidity and facilitates protonation by the bulk solvent. We define substrate neutralization by proton transfer for transport via a hydrophobic transport path as a general theme of the Amt/Mep/Rh ammonium and formate–nitrite transporters.     

Photosynthetic electron transport and establishment of an associated trans-thylakoid proton electrochemical gradient during development of the wheat leaf

Abstract Photosynthetic electron transport activities and the ability to generate and maintain a trans-thylakoid proton electrochemical gradient were examined during chloroplast development in 4-day-old wheat leaves grown under a diurnal light regime. Polarographic and spectropholometric studies on leaf tissue demonstrated that poorly developed chloroplasls at the leaf base could photo-oxidize water and transfer electrons from photosystem 2 to photosystem 1. The capacity for non-cyclic whole-chain electron transport increased during chloroplast development. Thylakoids isolated from the leaf base, although capable of pumping protons into the inlrathylakoid space, could not maintain a trans-membrane proton electrochemical gradient; this ability developed at later stages of chloroplast biogenesis in the leaf. The implications of these results for the energetics of the developing leaf are discussed.     

Oxygen and proton pathways in cytochrome c oxidase

Cytochrome c oxidase is a redox-driven proton pump, which couples the reduction of oxygen to water to the translocation of protons across the membrane. The recently solved x-ray structures of cytochrome c oxidase permit molecular dynamics simulations of the underlying transport processes. To eventually establish the proton pump mechanism, we investigate the transport of the substrates, oxygen and protons, through the enzyme. Molecular dynamics simulations of oxygen diffusion through the protein reveal a well-defined pathway to the oxygen-binding site starting at a hydrophobic cavity near the membrane-exposed surface of subunit I, close to the interface to subunit III. A large number of water sites are predicted within the protein, which could play an essential role for the transfer of protons in cytochrome c oxidase. The water molecules form two channels along which protons can enter from the cytoplasmic (matrix) side of the protein and reach the binuclear center. A possible pumping mechanism is proposed that involves a shuttling motion of a glutamic acid side chain, which could then transfer a proton to a propionate group of heme α₃. Proteins 30:100–107, 1998. © 1998 Wiley-Liss, Inc.     

The mechanism of proton exclusion in the aquaporin-1 water channel

Aquaporins are efficient, yet strictly selective water channels. Remarkably, proton permeation is fully blocked, in contrast to most other water-filled pores which are known to conduct protons well. Blocking of protons by aquaporins is essential to maintain the electrochemical gradient across cellular and subcellular membranes. We studied the mechanism of proton exclusion in aquaporin-1 by multiple non-equilibrium molecular dynamics simulations that also allow proton transfer reactions. From the simulations, an effective free energy profile for the proton motion along the channel was determined with a maximum-likelihood approach. The results indicate that the main barrier is not, as had previously been speculated, caused by the interruption of the hydrogen-bonded water chain, but rather by an electrostatic field centered around the fingerprint Asn-Pro-Ala (NPA) motif. Hydrogen bond interruption only forms a secondary barrier located at the ar/R constriction region. The calculated main barrier height of 25-30 kJ mol(-1) matches the barrier height for the passage of protons across pure lipid bilayers and, therefore, suffices to prevent major leakage of protons through aquaporins. Conventional molecular dynamics simulations additionally showed that negatively charged hydroxide ions are prevented from being trapped within the NPA region by two adjacent electrostatic barriers of opposite polarity.     

Local dynamics and structure of the solvated hydroxide ion in water

Details of the structural diffusion mechanism in proton transfer (PT) reactions involving a hydroxide ion in water and the structure of the solvated ions and transfer intermediates are in dispute. Here, we elucidate the mechanism of PT involving a hydroxide ion in water by molecular dynamics simulations using a dissociating water model based on ab initio calculations. We find that the hydroxide ion in bulk water is present as the four-coordinated OH^? (H₂O)₄ complex, which loses a water molecule before a PT occurs through the formation of proton sharing intermediates in general agreement with the previously disputed first principles studies of small systems.     

Solvent-assisted excited state proton transfer and photoacidity of 2-hydroxypyridine. A nonadiabatic dynamics study

Reactions involving proton transfer, especially those taking place in excited states (ESPT), received considerable attention. These reactions underlie many biological and biochemical processes that are vital to life. DNA mutations excited state funnelling and transport phenomena are just examples. Among many molecules undergoing this type of photoreactions, phenols show a unique property, the so-called photoacidity. In the present communication, solvent (methanol and ammonia) assisted excited state proton transfer and photo acidity of 2-hydroxypyridine (2HP) are investigated at the DFT M06-2X / def2pvv level of theory. Excited states of 2HP and its complexes with methanol and ammonia were examined at two levels. First, sampling a Wigner distribution of 300 points, the photoabsorption spectra were simulated within the nuclear ensemble approximation. Second, simulation of the dynamics of the excited states with the surface hopping method. Several separate spectral windows with three hundred trajectories each, were considered. Lifetime of excited states and photochemical channels observed were discussed.     

Metal Fluoride Inhibition of a P-type H+ Pump: STABILIZATION OF THE PHOSPHOENZYME INTERMEDIATE CONTRIBUTES TO POST-TRANSLATIONAL PUMP ACTIVATION*

The plasma membrane H⁺-ATPase is a P-type ATPase responsible for establishing electrochemical gradients across the plasma membrane in fungi and plants. This essential proton pump exists in two activity states: an autoinhibited basal state with a low turnover rate and a low H⁺/ATP coupling ratio and an activated state in which ATP hydrolysis is tightly coupled to proton transport. Here we characterize metal fluorides as inhibitors of the fungal enzyme in both states. In contrast to findings for other P-type ATPases, inhibition of the plasma membrane H⁺-ATPase by metal fluorides was partly reversible, and the stability of the inhibition varied with the activation state. Thus, the stability of the ATPase inhibitor complex decreased significantly when the pump transitioned from the activated to the basal state, particularly when using beryllium fluoride, which mimics the bound phosphate in the E2P conformational state. Taken together, our results indicate that the phosphate bond of the phosphoenzyme intermediate of H⁺-ATPases is labile in the basal state, which may provide an explanation for the low H⁺/ATP coupling ratio of these pumps in the basal state.     

Cyclic electron transfer in photosystem II in the marine diatom Phaeodactylum tricornutum

In Phaeodactylum tricornutum Photosystem II is unusually resistant to damage by exposure to high light intensities. Not only is the capacity to dissipate excess excitations in the antenna much larger and induced more rapidly than in other organisms, but in addition an electron transfer cycle in the reaction center appears to prevent oxidative damage when secondary electron transport cannot keep up with the rate of charge separations. Such cyclic electron transfer had been inferred from oxygen measurements suggesting that some of its intermediates can be reduced in the dark and can subsequently compete with water as an electron donor to Photosystem II upon illumination. Here, the proposed activation of cyclic electron transfer by illumination is confirmed and shown to require only a second. On the other hand the dark reduction of its intermediates, specifically of tyrosine Y_D, the only Photosystem II component known to compete with water oxidation, is ruled out. It appears that the cyclic electron transfer pathway can be fully opened by reduction of the plastoquinone pool in the dark. Oxygen evolution reappears after partial oxidation of the pool by Photosystem I, but the pool itself is not involved in cyclic electron transfer.     

Photochemistry and Photoinduced Proton-Transfer by Pharaonis Phoborhodopsin

Phoborhodopsin (pR or sensory rhodopsin II, sRII) is a photoreceptor of the negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. The photocycle of ppR is essentially as follows: ppR(498) ppR_K(540) ppR_KL(512) ppR_L(488) ppR_M(390) ppR_O(560) ppR (numbers in parenthesis denote the maximum absorbance). The photocycle is very similar to that of bacteriorhodopsin, but the rate of initial pigment recovery is about two-orders of magnitude slower. By low-temperature spectroscopy, two K-intermediates were found but the L intermediate was not detected. The lack of L indicates extraordinary stability of K at low temperature. ppR_M is photoactive similar to M of bR. The ground state ppR contains only all-trans retinal whereas ppR_M and ppR_O contain 13-cis and all-trans, respectively. ppR has the ability of lightinduced proton transport from the inside to the outside. Proton uptake occurs at the formation of ppR_O and the release at its decay. ppR associates with its transducer and this complex transmits a signal to the cytoplasm. The proton transport ability is lost when the complex forms, but the proton uptake and release still occur, suggesting that the proton movement is non-electrogenic (release and uptake occur from the same side). The stoichiometry of the complex between ppR and the transducer is 1 : 1. ppR or pR has absorption maximum at 500 nm, which is blue-shifted from those of other archaeal rhodopsins. The molecular mechanism of this color regulation is not yet solved.     

Identification of proton transfer pathways in the X-ray crystal structure of the bacterial reaction center from Rhodobacter sphaeroides

Structural features that have important implications for the fundamental process of transmembrane proton transfer are examined in the recently published high resolution atomic structures of the reaction center (RC) from Rhodobacter sphaeroides in the dark adapted state (DQ_AQ_B) and the charged separated state (D⁺Q_AQ_B ^–); the latter is the active state for proton transfer to the semiquinone. The structures have been determined at 2.2 Å and 2.6 Å resolution, respectively, as reported by Stowell et al. (1997) [Science 276: 812–816]. Three possible proton transfer pathways (P1, P2, P3) consisting of water molecules and/or protonatable residues were identified which connect the Q_B binding region with the cytoplasmic exposed surface at Asp H224 & Asp M240 (P1), Tyr M3 (P2) and Asp M17 (P3). All three represent possible pathways for proton transfer into the RC. P1 contains an uninterrupted chain of water molecules. This path could, in addition, facilitate the exchange of quinone for quinol during the photocycle by allowing water to move into and out of the binding pocket. Located near these pathways is a cluster of electrostatically interacting acid residues (Asp-L213, Glu-H173, Asp-M17, Asp H124, Asp-L210 and Asp H170) each being within 4.5 Å of a neighboring carboxylic acid or a bridging water molecule. This cluster could serve as an internal proton reservoir facilitating fast protonation of Q_B ^– that could occur at a rate greater than that attainable by proton uptake from solution.     

Internal water molecules and H-bonding in biological macromolecules: a review of structural features with functional implications.

Conserved structural patterns of internal water molecules and/or H-bond chains were observed and are here correlated in this review, which then describes two functional properties: equilibration of hydrostatic pressure and proton transport. Available evidence in support of these hypotheses is presented, together with suggested experiments to test them. High-resolution crystal structures of a variety of proteins were studied with interactive computer graphics. Conserved H-bonding linkages may be used as a paradigm for a rationalization of proton transport in membranes. The concept of the "proton wire," which links buried active-site amino acids with the surface of the protein raises the more general question of the functional role of the various molecular components.     

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.     

Photoelectrochemical Cycle of Bacteriorhodopsin

The scheme of the bacteriorhodopsin photocycle associated with a transmembrane proton transfer and electrogenesis is considered. The role of conformational changes in the polypeptide chain during the proton transport is discussed.     

Yeast protein–surfactant complexes uncouple microbial electron transfer and increase transmembrane leak of protons

Aims:  To explore the combined effect of yeast proteins and surfactants on bacterial metabolism.
Methods and Results:  Protein-rich cell-free supernatant from heat-shocked yeast Saccharomyces cerevisiae was combined with certain synthetic surfactants. These blends affected the metabolism of a Polyseed inoculum of aerobic bacteria, accelerating CO₂ production and consumption of nutrients from a sterile nutrient broth solution, without a concomitant accumulation of biomass. It is suggested that in the presence of the yeast protein–surfactant complexes, bacterial electron transport is uncoupled from biomass accumulation. The 'uncoupling hypothesis' is supported by experiments with model membranes, in which the same complexes induced proton leak similar to standard chemical uncouplers, such as dinitrophenol, indicating that uncoupling may occur at the stage of generation of the transmembrane pH gradient as the driving force for ATP production.
Conclusions:  Yeast protein–surfactant complexes behave as uncouplers of oxidative metabolism in bacteria and appear to do so by increasing proton permeability of membranes.
Significance and Impact of the Study:  Yeast proteins may be of interest as nontoxic, environmentally benign and economically sound agents accelerating oxidative bacterial metabolism while uncoupling it from biomass accumulation. There are actual and potential implications in waste water/soil decontamination, degreasing and other environmental technologies.     

Chance and design--proton transfer in water, channels and bioenergetic proteins

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.     

Proton transfer along the external proton channel of bacteriorhodopsin

The process of proton transfer along a proton channel is considered using bacteriorhodopsin as a model system, for which a large body of experimental data is available. The possible amino acid composition of the external proton half-channel of bacteriorhodopsin and the stepwise scheme of proton transfer consistent with experimental data are proposed. The rate of proton transfer between fixed centers is assessed for certain regions of this channel for which spectroscopic data are available.     

Structural and Functional Analysis of SoPIP2;1 Mutants Adds Insight into Plant Aquaporin Gating

Plant plasma membrane aquaporins facilitate water flux into and out of plant cells, thus coupling their cellular function to basic aspects of plant physiology. Posttranslational modifications of conserved phosphorylation sites, changes in cytoplasmic pH and the binding of Ca²⁺ can regulate water transport activity by gating the plasma membrane aquaporins. A structural mechanism unifying these diverse biochemical signals has emerged for the spinach aquaporin SoPIP2;1, although several questions concerning the opening mechanism remain. Here, we describe the X-ray structures of the S115E and S274E single SoPIP2;1 mutants and the corresponding double mutant. Phosphorylation of these serines is believed to increase water transport activity of SoPIP2;1 by opening the channel. However, all mutants crystallised in a closed conformation, as confirmed by water transport assays, implying that neither substitution fully mimics the phosphorylated state. Nevertheless, a half-turn extension of transmembrane helix 1 occurs upon the substitution of Ser115, which draws the C^α atom of Glu31 10 Å away from its wild-type conformation, thereby disrupting the divalent cation binding site involved in the gating mechanism. Mutation of Ser274 disorders the C-terminus but no other significant conformational changes are observed. Inspection of the hydrogen-bond interactions within loop D suggested that the phosphorylation of Ser188 may also produce an open channel, and this was supported by an increased water transport activity for the S188E mutant and molecular dynamics simulations. These findings add additional insight into the general mechanism of plant aquaporin gating.     

Exploring emergent properties in cellular homeostasis using OnGuard to model K and other ion transport in guard cells

It is widely recognized that the nature and characteristics of transport across eukaryotic membranes are so complex as to defy intuitive understanding. In these circumstances, quantitative mathematical modeling is an essential tool, both to integrate detailed knowledge of individual transporters and to extract the properties emergent from their interactions. As the first, fully integrated and quantitative modeling environment for the study of ion transport dynamics in a plant cell, OnGuard offers a unique tool for exploring homeostatic properties emerging from the interactions of ion transport, both at the plasma membrane and tonoplast in the guard cell. OnGuard has already yielded detail sufficient to guide phenotypic and mutational studies, and it represents a key step toward ‘reverse engineering’ of stomatal guard cell physiology, based on rational design and testing in simulation, to improve water use efficiency and carbon assimilation. Its construction from the HoTSig libraries enables translation of the software to other cell types, including growing root hairs and pollen. The problems inherent to transport are nonetheless challenging, and are compounded for those unfamiliar with conceptual ‘mindset’ of the modeler. Here we set out guidelines for the use of OnGuard and outline a standardized approach that will enable users to advance quickly to its application both in the classroom and laboratory. We also highlight the uncanny and emergent property of OnGuard models to reproduce the ‘communication’ evident between the plasma membrane and tonoplast of the guard cell.