Functions of the hydrophilic channels in protonmotive cytochrome c oxidase.
ABSTRACT: The structures and functions of hydrophilic channels in electron-transferring membrane proteins are discussed. A distinction is made between proton channels that can conduct protons and dielectric channels that are non-conducting but can dielectrically polarize in response to the introduction of charge changes in buried functional centres. Functions of the K, D and H channels found in A1-type cytochrome c oxidases are reviewed in relation to these ideas. Possible control of function by dielectric channels and their evolutionary relation to proton channels is explored.
Project description: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.
Project description:The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains an oxygen reductase, which uses HiPIP (high potential iron-sulfur protein) as an electron donor. The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I, III, and IV (named rcoxA to rcoxD) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstream of rcoxD. Examination of the amino acid sequence of the Rcox subunits shows that the subunits of the R. marinus enzyme have homology to the corresponding subunits of oxidases belonging to the superfamily of heme-copper oxidases. RcoxB has the conserved histidines involved in binding the binuclear center and the low-spin heme. All of the residues proposed to be involved in proton transfer channels are conserved, with the exception of the key glutamate residue of the D-channel (E(278), Paracoccus denitrificans numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine (Y) residue and the hydroxyl group of the following serine (S) may functionally substitute the glutamate carboxyl in proton transfer. RcoxA has an additional sequence for heme C binding, after the Cu(A) domain, that is characteristic of caa(3) oxidases belonging to the superfamily. Homology modeling of the structure of this cytochrome domain of subunit II shows no marked electrostatic character, especially around the heme edge region, suggesting that the interaction with a redox partner is not of an electrostatic nature. This observation is analyzed in relation to the electron donor for this caa(3) oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which uses an iron-sulfur protein as an electron donor, is structurally related to the caa(3) class of heme-copper cytochrome c oxidases. The data are discussed in the framework of the evolution of oxidases within the superfamily of heme-copper oxidases.
Project description:Respiratory complex I, NADH:ubiquinone oxidoreductase, is a large and complex integral membrane enzyme found in respiring bacteria and mitochondria. It is responsible in part for generating the proton gradient necessary for ATP production. Complex I serves as both a proton pump and an entry point for electrons into the respiratory chain. Although complex I is one of the most important of the respiratory complexes, it is also one of the least understood, with detailed structural information only recently available. In this study, full-finite-difference Poisson-Boltzmann calculations of the protonation state of respiratory complex I in various redox states are presented. Since complex I couples the oxidation and reduction of the NADH/ubiquinone redox couple to proton translocation, the interaction of the protonation and redox states of the enzyme are of the utmost significance. Various aspects of complex I function are presented, including the redox-Bohr effect, intercofactor interactions, and the effects of both the protein dielectric and inclusion of the membrane.
Project description:Heme-copper oxidases are membrane proteins found in the respiratory chain of aerobic organisms. They are the terminal electron acceptors coupling the translocation of protons across the membrane with the reduction of oxygen to water. Because the catalytic process occurs in the heme cofactors positioned well inside the protein matrix, proton channels must exist. However, due to the high structural divergence among this kind of proteins, the proton channels previously described are not necessarily conserved. In this work we modeled the structure of the quinol oxidase from Acidianus ambivalens using comparative modeling techniques for identifying proton channels. Additionally, given the high importance that water molecules may have in this process, we have developed a methodology, within the context of comparative modeling, to identify high water probability zones and to deconvolute them into chains of ordered water molecules. From our results, and from the existent information from other proteins from the same superfamily, we were able to suggest three possible proton channels: one K-, one D-, and one Q-spatial homologous proton channels. This methodology can be applied to other systems where water molecules are important for their biological function.
Project description:A mechanism for proton pumping is described that is based on chemiosmotic principles and the detailed molecular structures now available for cytochrome oxidases. The importance of conserved water positions and a step-wise gated process of proton translocation is emphasized, where discrete electron transfer events are coupled to proton uptake and expulsion. The trajectory of each pumped proton is the same for all four substrate electrons. An essential role for the His-Tyr cross-linked species is discussed, in gating of the D- and K-channels and as an acceptor/donor of electrons and protons at the binuclear center.
Project description:Nitric oxide reductases (NORs) are membrane proteins that catalyze the reduction of nitric oxide (NO) to nitrous oxide (N(2)O), which is a critical step of the nitrate respiration process in denitrifying bacteria. Using the recently determined first crystal structure of the cytochrome c-dependent NOR (cNOR) [Hino T, Matsumoto Y, Nagano S, Sugimoto H, Fukumori Y, et al. (2010) Structural basis of biological N2O generation by bacterial nitric oxide reductase. Science 330: 1666-70.], we performed extensive all-atom molecular dynamics (MD) simulations of cNOR within an explicit membrane/solvent environment to fully characterize water distribution and dynamics as well as hydrogen-bonded networks inside the protein, yielding the atomic details of functionally important proton channels. Simulations reveal two possible proton transfer pathways leading from the periplasm to the active site, while no pathways from the cytoplasmic side were found, consistently with the experimental observations that cNOR is not a proton pump. One of the pathways, which was newly identified in the MD simulation, is blocked in the crystal structure and requires small structural rearrangements to allow for water channel formation. That pathway is equivalent to the functional periplasmic cavity postulated in cbb(3) oxidase, which illustrates that the two enzymes share some elements of the proton transfer mechanisms and confirms a close evolutionary relation between NORs and C-type oxidases. Several mechanisms of the critical proton transfer steps near the catalytic center are proposed.
Project description:In the past few decades, gate insulators with a high dielectric constant (high-k dielectric) enabling a physically thick but dielectrically thin insulating layer, have been used to replace traditional SiOx insulator and to ensure continuous downscaling of Si-based transistor technology. However, due to the non-silicon derivative natures of the high-k metal oxides, transport properties in these dielectrics are still limited by various structural defects on the hetero-interfaces and inside the dielectrics. Here, we show that another insulating silicon compound, amorphous silicon nitride (a-Si3N4), is a promising candidate of effective electrical insulator for use as a high-k dielectric. We have examined a-Si3N4 deposited using the plasma-assisted atomic beam deposition (PA-ABD) technique in an ultra-high vacuum (UHV) environment and demonstrated the absence of defect-related luminescence; it was also found that the electronic structure across the a-Si3N4/Si heterojunction approaches the intrinsic limit, which exhibits large band gap energy and valence band offset. We demonstrate that charge transport properties in the metal/a-Si3N4/Si (MNS) structures approach defect-free limits with a large breakdown field and a low leakage current. Using PA-ABD, our results suggest a general strategy to markedly improve the performance of gate dielectric using a nearly defect-free insulator.
Project description:Voltage-gated proton channels are increasingly implicated in cellular proton homeostasis. Proton currents were originally identified in snail neurons less than 40 years ago, and subsequently shown to play an important auxiliary role in the functioning of reactive oxygen species (ROS)-generating nicotinamide adenine dinucleotide phosphate (NADPH) oxidases. Molecular identification of voltage-gated proton channels was achieved less than 10 years ago. Interestingly, so far, only one gene coding for voltage-gated proton channels has been identified, namely hydrogen voltage-gated channel 1 (HVCN1), which codes for the HV1 proton channel protein. Over the last years, the first picture of putative physiological functions of HV1 has been emerging.The best-studied role remains charge and pH compensation during the respiratory burst of the phagocyte NADPH oxidase (NOX). Strong evidence for a role of HV1 is also emerging in sperm biology, but the relationship with the sperm NOX5 remains unclear. Probably in many instances, HV1 functions independently of NOX: for example in snail neurons, basophils, osteoclasts, and cancer cells.Generally, ion channels are good drug targets; however, this feature has so far not been exploited for HV1, and hitherto no inhibitors compatible with clinical use exist. However, there are emerging indications for HV1 inhibitors, ranging from diseases with a strong activation of the phagocyte NOX (e.g., stroke) to infertility, osteoporosis, and cancer.Clinically useful HV1-active drugs should be developed and might become interesting drugs of the future.
Project description:Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis. In order to provide an experimental means to address this issue, FTIR spectroscopy in the 3,560-3,800 cm(-1) range has been used to detect weakly H-bonded water molecules in bovine cytochrome c oxidase that might change during catalysis. Full redox spectra exhibited at least four signals at 3,674(+), 3,638(+), 3,620(-), and 3,607(+) cm(-1). A more complex set of signals was observed in spectra of photolysis of the ferrous-CO compound, a reaction that mimics the catalytic oxygen binding step, and their D(2)O and H(2)(18)O sensitivities confirmed that they arose from water molecule rearrangements. Fitting with Gaussian components indicated the involvement of up to eight waters in the photolysis transition. Similar signals were also observed in photolysis spectra of the ferrous-CO compound of bacterial CcO from Paracoccus denitrificans. Such water changes are discussed in relation to roles in hydrophilic channels and proton/electron coupling mechanism.
Project description:The synthesis of high-conductivity solid-state electrolyte materials with eliminated polarization loss is a great challenge. Here we show a promising potential of single-ion block copolymers with crystalline protogenic channels as efficient proton conductors. Through the self-organization of zwitterion, imidazole, and polystyrene sulfonate with controlled dipolar interactions therein, the distance between neighboring proton donors and acceptors in ionic crystals, as well as the dipolar orientation in nanoscale ionic phases was precisely tuned. This allowed a markedly high static dielectric constant comparable to water and fast structural diffusion of protons with a low potential barrier for single-ion polymers. The optimized sample exhibited a high proton diffusion coefficient of 2.4?×?10-6?cm2?s-1 under anhydrous conditions at 90?°C.