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PubMed1991

The possible role of redox-associated protons in growth of plant cells.

The protons excreted by plant cells may arise by two different mechanisms: (1) by the action of the plasma membrane H(+)-ATPase and (2) by plasma membrane redox reactions. The exact proportion from each source is not known, but the plasma membrane H(+)-ATPase is, by far, the major contributor to proton efflux. There is still some questions of whether the redox-associated protons produced by NADH oxidation on the inner side of the plasma membrane traverse the membrane in a 1:1 relationship with electrons generated in the redox reactions. Membrane depolarization observed in the presence of ferricyanide reduction by plasma membranes of whole cells or tissues or the lag period between ferricyanide reduction and medium acidification argue that only scalar protons may be involved. The other major argument against tight coupling between protons and electrons involves the concept of strong charge compensation. When ferricyanide is reduced to ferrocyanide on the outside of cells or tissues, an extra negative charge arises, which is compensated for by the release of H+ or K+, so that the total ratio of increased H+ plus K+ equals the electrons transferred by transmembrane electron transport. These are strong arguments against a tight coupling between electrons and protons excreted by the plasma membrane. On the other hand, there is no question that inhibitor studies provide evidence for two mechanisms of proton generation by plasma membranes. When the H(+)-ATPase activity is totally inhibited, the addition of ferricyanide induces a burst of extra proton excretion, or vice versa, when plasma membrane redox reactions are inhibited, the H(+)-ATPase can function normally. Since plasma membrane redox reactions and associated H+ excretion are related to growth it is possible that in plants the ATPase-generated protons have a different function from redox-associated protons. The H(+)-ATPase-generated protons have been considered for many years to be necessary for cell wall expansion, allowing elongation to take place. A special function of the redox-generated protons may be in initiating proliferative cell growth, based on the presence of a hormone-stimulated NADH oxidase in membranes of soybean hypocotyls and stimulation of root growth by low concentrations of oxidants. Here we propose that this NADH oxidase and the redox protons released by its action control growth. The mechanism for this may be the evolution of protons into a special membrane domain, from which a signal to initiate cell proliferation may originate, independent of the action of the H(+)-ATPase-generated protons.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Division↗
PubMed2003

Redox-driven proton pumping by heme-copper oxidases.

One of the key problems of molecular bioenergetics is the understanding of the function of redox-driven proton pumps on a molecular level. One such class of proton pumps are the heme-copper oxidases. These enzymes are integral membrane proteins in which proton translocation across the membrane is driven by electron transfer from a low-potential donor, such as, e.g. cytochrome c, to a high-potential acceptor, O(2). Proton pumping is associated with distinct exergonic reaction steps that involve gradual reduction of oxygen to water. During the process of O(2) reduction, unprotonated high pK(a) proton acceptors are created at the catalytic site. Initially, these proton acceptors become protonated as a result of intramolecular proton transfer from a residue(s) located in the membrane-spanning part of the enzyme, but removed from the catalytic site. This residue is then reprotonated from the bulk solution. In cytochrome c oxidase from Rhodobacter sphaeroides, the proton is initially transferred from a glutamate, E(I-286), which has an apparent pK(a) of 9.4. According to a recently published structure of the enzyme, the deprotonation of E(I-286) is likely to result in minor structural changes that propagate to protonatable groups on the proton output (positive) side of the protein. We propose that in this way, the free energy available from the O(2) reduction is conserved during the proton transfer. On the basis of the observation of these structural changes, a possible proton-pumping model is presented in this paper. Initially, the structural changes associated with deprotonation of E(I-286) result in the transfer of a proton to an acceptor for pumped protons from the input (negative) side of the membrane. After reprotonation of E(I-286) this acceptor releases a proton to the output side of the membrane.

Copper↗
PubMed2002

Proton transfer dynamics on the surface of the late M state of bacteriorhodopsin.

The cytoplasmic surface of the BR (initial) state of bacteriorhodopsin is characterized by a cluster of three carboxylates that function as a proton-collecting antenna. Systematic replacement of most of the surface carboxylates indicated that the cluster is made of D104, E161, and E234 (Checover, S., Y. Marantz, E. Nachliel, M. Gutman, M. Pfeiffer, J. Tittor, D. Oesterhelt, and N. Dencher. 2001. Biochemistry. 40:4281-4292), yet the BR state is a resting configuration; thus, its proton-collecting antenna can only indicate the presence of its role in the photo-intermediates where the protein is re-protonated by protons coming from the cytoplasmic matrix. In the present study we used the D96N and the triple (D96G/F171C/F219L) mutant for monitoring the proton-collecting properties of the protein in its late M state. The protein was maintained in a steady M state by continuous illumination and subjected to reversible pulse protonation caused by repeated excitation of pyranine present in the reaction mixture. The re-protonation dynamics of the pyranine anion was subjected to kinetic analysis, and the rate constants of the reaction of free protons with the surface groups and the proton exchange reactions between them were calculated. The reconstruction of the experimental signal indicated that the late M state of bacteriorhodopsin exhibits an efficient mechanism of proton delivery to the unoccupied-most basic-residue on its cytoplasmic surface (D38), which exceeds that of the BR configuration of the protein. The kinetic analysis was carried out in conjunction with the published structure of the M state (Sass, H., G. Büldt, R. Gessenich, D. Hehn, D. Neff, R. Schlesinger, J. Berendzen, and P. Ormos. 2000. Nature. 406:649-653), the model that resolves most of the cytoplasmic surface. The combination of the kinetic analysis and the structural information led to identification of two proton-conducting tracks on the protein's surface that are funneling protons to D38. One track is made of the carboxylate moieties of residues D36 and E237, while the other is made of D102 and E232. In the late M state the carboxylates of both tracks are closer to D38 than in the BR (initial) state, accounting for a more efficient proton equilibration between the bulk and the protein's proton entrance channel. The triple mutant resembles in the kinetic properties of its proton conducting surface more the BR-M state than the initial state confirming structural similarities with the BR-M state and differences to the BR initial state.

Bacteriorhodopsins↗
PubMed1985

Kinetic analysis of protonation of a specific site on a buffered surface of a macromolecular body.

The kinetics of protonation of a specific site on a macromolecular structure (micelle) in buffered solution was studied with the purpose of evaluating the effect of buffer on the observed dynamics. The experimental system consisted of the following elements: Brij 58 micelles serving as homogeneous uncharged macromolecular bodies, bromocresol green, a well-adsorbed proton detector, and 2-naphthol-3,6-disulfonate as a proton emitter in the bulk. Imidazole was the mobile buffer while neutral red, which has a high affinity for the micellar surface, served as the immobile buffer. An intensive laser pulse ejects a proton from the proton emitter, and the subsequent proton-transfer reactions are measured by fast spectrophotometric methods. The dynamics of proton pulse in buffered solution are characterized by a very rapid trapping of the discharged protons by the abundant buffer molecules. This event has a major effect on the kinetic regime of the reaction. During the first 200 ns the proton flux is rate limited by free-proton diffusion. After this period, when the free-proton concentration decayed to the equilibrium level, the relaxation of the system is carried out by the diffusion of buffer. Thus in the buffered biochemical system, at neutral pH, most of proton flux between active sites and bulk is carried out by buffer molecules--not by diffusion of free protons. Surface groups on a high molecular weight body exchange protons among them at a very fast rate. This reaction has a major role on proton transfer from a specific site to the bulk.(ABSTRACT TRUNCATED AT 250 WORDS)

Binding Sites↗
PubMed1987

Conductance routes for protons across membrane barriers.

Simple phospholipid bilayers show a high level of permeability to protons; in spite of this fact, large proton gradients existing across such bilayers may decay very slowly. In sealed systems, the free movement of protons across a membrane barrier is severely restricted by the coincident development of a proton diffusion potential. Using the fluorescent weak acid N-[5-(dimethylamino)naphth-1-ylsulfonyl]glycine strongly buffered systems movement of the small number of protons giving rise to this electrical potential is insufficient to perturb the proton concentration gradient; significant flux of protons (and hence significant collapse of the concentration gradient) can only occur if protons traverse the membrane as part of an electroneutral complex or if there is a balancing flow of appropriate counterions. In both instances, proton flux is obligatorily coupled to the translocation of species other than protons. In weakly buffered systems, the small initial uncoupled electrogenic flux of protons may significantly alter the concentration gradient. This initial rapid gradient collapse caused by uncoupled electrogenic proton movements is then superimposed upon the residual collapse attributable to tightly coupled proton flux. The initial uncoupled electrogenic proton flux shows a temperature dependence very similar to that demonstrated for water permeation across simple lipid bilayers; upon cooling, there is a sharp decrease in flux at the temperature coinciding with the main gel-liquid-crystalline phase transition of the lipid. The coupled proton flux shows a markedly different temperature dependence with no dramatic change in rate at the phase transition temperature and strong similarity to the behavior previously seen with solutes known to be permeating as electrically neutral compounds.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid Bilayers↗
PubMed2006

TPD-based evaluation of near threshold mono-energetic proton energies for the (7)Li(p,n)(7)Be production of neutrons for BNCT.

An evaluation of mono-energetic proton energies ranging from 1.885 MeV to 1.920 MeV was carried out to determine the viability of these near threshold energies in producing neutrons for BNCT via the (7)Li(p,n)(7)Be reaction. Neutron fields generated at these proton energies were assessed using the treatable protocol depth (TPD) and the maximum TPD (TPD(max)) as evaluation indices. The heavy charged particle (HCP) dose rate to tumour was likewise applied as a figure of merit in order to account for irradiation time and required proton current. Incident proton energies closer to the reaction threshold generated deeper TPDs compared to higher energy protons when no boron dose enhancers (BDE) were placed in the irradiation field. Introducing a BDE resulted in improved TPDs for high proton energies but their achievable TPD(max) were comparatively lower than that obtained for lower proton energies. In terms of the HCP dose rate to tumour, higher proton energies generated neutron fields that yielded higher dose rates both at TPD(max) and at fixed depths of comparison. This infers that higher currents are required to deliver the prescribed treatment dose to tumours for proton beams with energies closer to the (7)Li(p,n)(7)Be reaction threshold and more achievable proton currents of around 10 mA or less for proton energies from 1.900 MeV and above. The dependence on incident proton energy of the TPD, TPD(max) and the HCP dose rate to tumour with respect to the (10)B concentration in tumour and healthy tissues were also clarified in this study. Increasing the (10)B concentration in tumour while maintaining a constant T/N ratio resulted in deeper TPD(max) where a greater change in TPD(max) was obtained for proton energies closer to the (7)Li(p,n)(7)Be reaction threshold. The HCP dose rates to tumour for all proton energies also went up, with the higher proton energies benefiting more from the increased (10)B concentration.

Beryllium↗
PubMed2006

Mitochondrial proton leak rates in the slow, oxidative myotomal muscle and liver of the endothermic shortfin mako shark (Isurus oxyrinchus) and the ectothermic blue shark (Prionace glauca) and leopard shark (Triakis semifasciata).

Mitochondrial proton leak was assessed as a potential heat source in the slow, oxidative (red) locomotor muscle and liver of the shortfin mako shark (Isurus oxyrinchus), a regional endotherm that maintains the temperature of both tissues elevated above ambient seawater temperature. We hypothesized that basal proton leak rates in red muscle and liver mitochondria of the endothermic shortfin mako shark would be greater than those of the ectothermic blue shark (Prionace glauca) and leopard shark (Triakis semifasciata). Respiration rate and membrane potential in isolated mitochondria were measured simultaneously at 20 degrees C using a Clark-type oxygen electrode and a lipophilic probe (triphenylmethylphosphonium, TPMP(+)). Succinate-stimulated respiration was titrated with inhibitors of the electron transport chain, and the non-linear relationship between respiration rate and membrane potential was quantified. Mitochondrial densities of both tissues were measured by applying the point-contact method to electron micrographs so that proton leak activity of the entire tissue could be assessed. In all three shark species, proton leak occurred at a higher rate in red muscle mitochondria than in liver mitochondria. For each tissue, the proton leak curves of the three species overlapped and, at a membrane potential of 160 mV, mitochondrial proton leak rate (nmol H(+) min(-1) mg(-1) protein) did not differ significantly between the endothermic and ectothermic sharks. This finding indicates that red muscle and liver mitochondria of the shortfin mako shark are not specialized for thermogenesis by having a higher proton conductance. However, mako mitochondria did have higher succinate-stimulated respiration rates and membrane potentials than those of the two ectothermic sharks. This means that under in vivo conditions mitochondrial proton leak rates may be higher in the mako than in the ectothermic species, due to greater electron transport activity and a larger proton gradient driving proton leak. We also estimated each tissue's total proton leak by combining mitochondrial proton leak rates at 160 mV and tissue mitochondrial density data with published values of relative liver or red muscle mass for each of the three species. In red muscle, total proton leak was not elevated in the mako shark relative to the two ectothermic species. In the liver, total proton leak would be higher in the mako shark than in both ectothermic species, due to a lower proton conductance in the blue shark and a lower liver mitochondrial content in the leopard shark, and thus may contribute to endothermy.

Animals↗
PubMed2001

Response of thyroid follicular cells to gamma irradiation compared to proton irradiation. I. Initial characterization of DNA damage, micronucleus formation, apoptosis, cell survival, and cell cycle phase redistribution.

The RBE of protons has been assumed to be equivalent to that of photons. The objective of this study was to determine whether radiation-induced DNA and chromosome damage, apoptosis, cell killing and cell cycling in organized epithelial cells was influenced by radiation quality. Thyroid-stimulating hormone-dependent Fischer rat thyroid cells, established as follicles, were exposed to gamma rays or proton beams delivered acutely over a range of physical doses. Gamma-irradiated cells were able to repair DNA damage relatively rapidly so that by 1 h postirradiation they had approximately 20% fewer exposed 3' ends than their counterparts that had been irradiated with proton beams. The persistence of free ends of DNA in the samples irradiated with the proton beam implies that either more initial breaks or a quantitatively different type of damage had occurred. These results were further supported by an increased frequency of chromosomal damage as measured by the presence of micronuclei. Proton-beam irradiation induced micronuclei at a rate of 2.4% per gray, which at 12 Gy translated to 40% more micronuclei than in comparable gamma-irradiated cultures. The higher rate of micronucleus formation and the presence of larger micronuclei in proton-irradiated cells was further evidence that a qualitatively more severe class of damage had been induced than was induced by gamma rays. Differences in the type of damage produced were detected in the apoptosis assay, wherein a significant lag in the induction of apoptosis occurred after gamma irradiation that did not occur with protons. The more immediate expression of apoptotic cells in the cultures irradiated with the proton beam suggests that the damage inflicted was more severe. Alternatively, the cell cycle checkpoint mechanisms required for recovery from such damage might not have been invoked. Differences based on radiation quality were also evident in the alpha components of cell survival curves (0.05 Gy(-1) for gamma rays, 0.12 Gy(-1) for protons), which suggests that the higher level of survival of gamma-irradiated cells could be attributed to the persistence of nonlethally irradiated thyrocytes and/or the capacity to repair damage more effectively than cells exposed to equal physical doses of protons. The final assessment in this study was radiation-induced cell cycle phase redistribution. Gamma rays and protons produced a similar dose-dependent redistribution toward a predominantly G(2)-phase population. From our cumulative results, it seems likely that a majority of the proton-irradiated cells would not continue to divide. In conclusion, these findings suggest that there are quantitative and qualitative differences in the biological effects of proton beams and gamma rays. These differences could be due to structured energy deposition from the tracks of primary protons and the associated high-LET secondary particles produced in the targets. The results suggest that a simple dose-equivalent approach to dosimetry may be inadequate to compare the biological responses of cells to photons and protons.

Animals↗
PubMed1999

Aspartate-132 in cytochrome c oxidase from Rhodobacter sphaeroides is involved in a two-step proton transfer during oxo-ferryl formation.

The aspartate-132 in subunit I (D(I-132)) of cytochrome c oxidase from Rhodobacter sphaeroides is located on the cytoplasmic surface of the protein at the entry point of a proton-transfer pathway used for both substrate and pumped protons (D-pathway). Replacement of D(I-132) by its nonprotonatable analogue asparagine (DN(I-132)) has been shown to result in a reduced overall activity of the enzyme and impaired proton pumping. The results from this study show that during oxidation of the fully reduced enzyme the reaction was inhibited after formation of the oxo-ferryl (F) intermediate (tau congruent with 120 microseconds). In contrast to the wild-type enzyme, in the mutant enzyme formation of this intermediate was not associated with proton uptake from solution, which is the reason the DN(I-132) enzyme does not pump protons. The proton needed to form F was presumably taken from a protonatable group in the D-pathway (e.g., E(I-286)), which indicates that in the wild-type enzyme the proton transfer during F formation takes place in two steps: proton transfer from the group in the pathway is followed by faster reprotonation from the bulk solution, through D(I-132). Unlike the wild-type enzyme, in which F formation is coupled to internal electron transfer from CuA to heme a, in the DN(I-132) enzyme this electron transfer was uncoupled from formation of the F intermediate, which presumably is due to the impaired charge-compensating proton uptake from solution. In the presence of arachidonic acid which has been shown to stimulate the turnover activity of the DN(I-132) enzyme (Fetter et al. (1996) FEBS Lett. 393, 155), proton uptake with a time constant of approximately 2 ms was observed. However, no proton uptake associated with formation of F (tau congruent with 120 micros) was observed, which indicates that arachidonic acid can replace the role of D(I-132), but it cannot transfer protons as fast as the Asp. The results from this study show that D(I-132) is crucial for efficient transfer of protons into the enzyme and that in the DN(I-132) mutant enzyme there is a "kinetic barrier" for proton transfer into the D-pathway.

Amino Acid Substitution↗
PubMed1983

Light-dependent proton efflux from chloroplast thylakoids.

This work clarifies certain aspects of proton conductivity estimates in the light compared to dark conditions for spinach chloroplast thylakoid membranes. A method is presented, with kinetic analysis to justify it, that permits the separation of the proton influx and efflux rate constants, the sum of which contributes to the measured apparent first-order rate constant for proton efflux linked to basal electron transport. Proton fluxes linked to ATP formation were not dealt with. Using this technique it was shown that dicyclohexylcarbodiimide, an inhibitor of proton channel function, completely blocks a component of proton efflux in the light, as well as partially blocking the proton efflux in the dark. Antibody against purified chloroplast coupling factor (CF1) inhibits the light-dependent proton efflux, but has no effect on the dark proton efflux. Those data are consistent with there being a proton efflux pathway through the coupling factor complex, both in the light and the dark. The H+ efflux through the coupling factor was closely correlated with adenine nucleotide exchange activity. As suggested by others, such exchange activity may be an indication of conformational changes linked to the activation of the coupling factor. A plausible model is that the positive proton electrochemical potential gradient leads to an interaction between protons and the coupling factor, causing a conformational change, which leads to adenine nucleotide exchange linked to the passage of protons through the coupling complex. The nucleotide exchange activity reflects a transition from a higher to a lower binding affinity. Some of the Gibbs free energy lost in the dissipation of the proton gradient must be conserved in the transition to the lower affinity adenine nucleotide binding form of the coupling factor protein complex.

Adenine Nucleotides↗
PubMed2006

Dehydration versus deamination of N-terminal glutamine in collision-induced dissociation of protonated peptides.

Some of the most prominent "neutral losses" in peptide ion fragmentation are the loss of ammonia and water from N-terminal glutamine. These processes are studied by electrospray ionization mass spectrometry in singly- and doubly-protonated peptide ions undergoing collision-induced dissociation in a triple quadrupole and in an ion trap instrument. For this study, four sets of peptides were synthesized: (1) QLLLPLLLK and similar peptides with K replaced by R, H, or L, and Q replaced by a number of amino acids, (2) QLnK (n = 0, 1, 3, 5, 7, 9, 11), (3) QLnR (n = 0, 1, 3, 5, 7, 9), and (4) QLn (n = 1, 2, 3, 4, 8). The results for QLLLPLLLK and QLLLPLLLR show that the singly protonated ions undergo loss of ammonia and to a smaller extent loss of water, whereas the doubly protonated ions undergo predominant loss of water. The fast fragmentation next to P (forming the y5 ion) occurs to a larger extent than the neutral losses from the singly protonated ions but much less than the water loss from the doubly protonated ions. The results from these and other peptides show that, in general, when N-terminal glutamine peptides have no "mobile protons", that is, the number of charges on the peptide is no greater than the number of basic amino acids (K, R, H), deamination is the predominant neutral loss fragmentation, but when mobile protons are present the predominant process is the loss of water. Both of these processes are faster than backbone fragmentation at the proline. These results are rationalized on the basis of resonance stabilization of the two types of five-membered ring products that would be formed in the neutral loss processes; the singly protonated ion yields the more stable neutral pyrrolidinone ring whereas the doubly protonated ion yields the protonated aminopyrroline ring (see Schemes). The generality of these trends is confirmed by analyzing an MS/MS spectra library of peptides derived from tryptic digests of yeast. In the absence of mobile protons, glutamine deamination is the most rapid neutral loss process. For peptides with mobile protons, dehydration from glutamine is far more rapid than from any other amino acid. Most strikingly, end terminal glutamine is by far the most labile source of neutral loss in excess-proton peptides, but not highly exceptional when mobile protons are not available. In addition, rates of deamination are faster in lysine versus arginine C-terminus peptides and 20 times faster in positively charged than negatively charged peptides, demonstrating that these formal neutral loss reactions are not "neutral reactions" but depend on charge state and stability.

Amino Acid Sequence↗
PubMed1998

Contribution of proton release to the B2 photocurrent of bacteriorhodopsin.

The contribution of proton release from the so-called proton release group to the microsecond B2 photocurrent from bacteriorhodopsin (bR) oriented in polyacrylamide gels was determined. The fraction of the B2 current due to proton release was resolved by titration of the proton release group in M. At pH values below the pKa of the proton release group in M, the proton release group cannot release its proton during the first half of the bacteriorhodopsin photocycle. At these pH values, the B2 photocurrent is due primarily to translocation of the Schiff base proton to Asp85. The B2 photocurrent was measured in wild-type bR gels at pH 4.5-7.5, in 100 mM KCl/50 mM phosphate. The B2 photocurrent area (proportional to the amount of charge moved) exhibits a pH dependence with a pKa of 6.1. This is suggested to be the pKa of the proton release group in M; the value obtained is in good agreement with previous results obtained by examining photocycle kinetics and pH-sensitive dye signals. In the mutant Glu204Gln, the B2 photocurrent of the mutant membranes was pH independent between pH 4 and 7. Because the proton release group is incapacitated, and early proton release is eliminated in the Glu204Gln mutant, this supports the idea that the pH dependence of the B2 photocurrent in the wild type reflects the titration of the proton release group. In wild-type bacteriorhodopsin, proton release contributes approximately half of the B2 area at pH 7.5. The B2 area in the Glu204Gln mutant is similar to that in the wild type at pH 4.5; in both cases, the B2 current is likely due only to movement of the Schiff base proton to Asp85.

Aspartic Acid↗
PubMed1998

Attenuation of proton currents by methanol in a dioxolane-linked gramicidin A channel in different lipid bilayers.

The mobility of protons in a dioxolane-linked gramicidin A channel (D1) is comparable to the mobility of protons in aqueous solutions (Cukierman, S., E. P. Quigley, and D. S. Crumrine. 1997. Biophys. J. 73:2489-2502). Aliphatic alcohols decrease the mobility of H+ in aqueous solutions. In this study, the effects of methanol on proton conduction through D1 channels were investigated in different lipid bilayers and at different HCl concentrations. Methanol attenuated H+ currents in a voltage-independent manner. Attenuation of proton currents was also independent of H+ concentrations in solution. In phospholipid bilayers, methanol decreased the single channel conductance to protons without affecting the binding affinity of protons to bilayers. In glycerylmonooleate membranes, the attenuation of single channel proton conductances qualitatively resembled the decrease of conductivities of HCl solutions by methanol. However, in both types of lipid bilayers, single channel proton conductances through D1 channels were considerably more attenuated than the conductivities of different HCl solutions. This suggests that methanol modulates single proton currents through D1 channels. It is proposed that, on average, one methanol molecule binds to a D1 channel, and attenuates H+ conductance. The Gibbs free energy of this process (DeltaG0) is approximately 1.2 kcal/mol, which is comparable to the free energy of decrease of HCl conductivity in methanol solutions (1.6 kcal/mol). Apolar substances like urea and glucose that do not transport protons in HCl solutions and do not permeate D1 channels decreased solution conductivity and single channel conductance by a considerably larger proportion than methanol. Cs+ currents through D1 channels were considerably less (fivefold) attenuated by methanol than proton currents. It is proposed that methanol partitions inside the pore of gramicidin channels and delays the transfer of protons between water and methanol molecules, causing a significant attenuation of the single channel proton conductance. Gramicidin channels offer an interesting experimental model to study proton hopping along a single chain of water molecules interrupted by a single methanol molecule.

Biophysical Phenomena↗
PubMed1983

Nature and locations of the most slowly exchanging peptide NH protons in residues 1 to 19 of ribonuclease S.

The locations have been found of the eight most slowly exchanging peptide protons in residues 1 to 19 of ribonuclease S. The resonance lines of these eight protons are resolved by proton magnetic resonance at 360 MHz when either S-peptide (residues 1 to 19) or peptide 1-15 is bound to S-protein (residues 21 to 124). Other peptide protons have been removed by exchange in the sample preparation [( 1H]S-peptide is added to deuterated S-protein in D2O), and also by exchange-out of the less protected protons in residues 1 to 19. At pH 5.1, 0 degrees C, there is a 100-fold difference in rates of exchange between the eight most protected protons and the less protected protons of S-peptide. The highly protected protons are protected 10(4)-fold compared to free S-peptide. The protected protons have been identified by 1H nuclear magnetic resonance after denaturing ribonuclease S in greater than or equal to 3 M-urea-d4, D2O, pH 2.3, -4 degrees C, followed by comparing the chemical shifts of the remaining eight protons with the known -NH spectrum of the free peptide, which has been assigned from the two-dimensional homonuclear correlated spectrum and by comparison with earlier work. The eight highly protected NH protons are localized in one segment, residues 7 to 14. All eight protons are H-bonded: those of residues 7 to 13 are H-bonded within the 3-13 alpha-helix and that of residue 14 is H-bonded to the beta-sheet. The NH proton of residue 16, which also is H-bonded to the beta-sheet, is not one of the highly protected protons. Both the N atoms of the eight NH groups and also the O atoms of their CO acceptor groups are shielded from solvent in most cases, according to the molecular area calculations of Finney (1978).

Amides↗
PubMed1981

Linear relations between proton current and pH gradient in bacteriorhodopsin liposomes.

The dependence of proton movement across the membrane of bacteriorhodopsin liposomes on the pH gradient was investigated. Under the appropriate experimental conditions, endogenous proton (or hydroxyl) leakage, proton movement catalyzed by protonophore or nigericin, and light-driven proton translocation depend linearly on the pH gradient. This justifies the use of linear proton flux vs. protonmotive force relations in a recent mosaic thermodynamic description of ion translocation in bacteriorhodopsin liposomes [Westerhoff, H. V., Scholte, B. J., & Hellingwerf, K. J. (1979) Biochim. Biophys. Acta 547, 544-560]. Since bacteriorhodopsin liposomes are a model system for all biological energy transducing systems in which proton pumps are involved, these findings also explain linear relations between proton flux and protonmotive force observed in and postulated for those systems. In cases where the membrane potential is not clamped at a low value, an initial phase of rapid proton movement occurs, followed by a phase of slower proton movement. The rate of proton movement during the slow phase is again linear with the pH gradient. Such a linear relation is not observed for the fast phase. Since the rapid proton movement phase is also observed in liposomes without bacteriorhodopsin, it is not due (only) to dissociation of scalar protons from bacteriorhodopsin. We suggest that during the initial phase of proton movement, the proton flux is not yet electrically compensated by the fluxes of other ions.

Bacteriorhodopsins↗
PubMed2007

A proposed proton shuttle mechanism for saccharopine dehydrogenase from Saccharomyces cerevisiae.

Saccharopine dehydrogenase [N6-(glutaryl-2)-L-lysine:NAD oxidoreductase (L-lysine forming)] catalyzes the final step in the alpha-aminoadipate pathway for lysine biosynthesis. It catalyzes the reversible pyridine nucleotide-dependent oxidative deamination of saccharopine to generate alpha-Kg and lysine using NAD+ as an oxidizing agent. The proton shuttle chemical mechanism is proposed on the basis of the pH dependence of kinetic parameters, dissociation constants for competitive inhibitors, and isotope effects. In the direction of lysine formation, once NAD+ and saccharopine bind, a group with a pKa of 6.2 accepts a proton from the secondary amine of saccharopine as it is oxidized. This protonated general base then does not participate in the reaction again until lysine is formed at the completion of the reaction. A general base with a pKa of 7.2 accepts a proton from H2O as it attacks the Schiff base carbon of saccharopine to form the carbinolamine intermediate. The same residue then serves as a general acid and donates a proton to the carbinolamine nitrogen to give the protonated carbinolamine. Collapse of the carbinolamine is then facilitated by the same group accepting a proton from the carbinolamine hydroxyl to generate alpha-Kg and lysine. The amine nitrogen is then protonated by the group that originally accepted a proton from the secondary amine of saccharopine, and products are released. In the reverse reaction direction, finite primary deuterium kinetic isotope effects were observed for all parameters with the exception of V2/K(NADH), consistent with a steady-state random mechanism and indicative of a contribution from hydride transfer to rate limitation. The pH dependence, as determined from the primary isotope effect on DV2 and D(V2/K(Lys)), suggests that a step other than hydride transfer becomes rate-limiting as the pH is increased. This step is likely protonation/deprotonation of the carbinolamine nitrogen formed as an intermediate in imine hydrolysis. The observed solvent isotope effect indicates that proton transfer also contributes to rate limitation. A concerted proton and hydride transfer is suggested by multiple substrate/solvent isotope effects, as well as a proton transfer in another step, likely hydrolysis of the carbinolamine. In agreement, dome-shaped proton inventories are observed for V2 and V2/K(Lys), suggesting that proton transfer exists in at least two sequential transition states.

Deuterium↗
PubMed1998

Energetics of electron-transfer and protonation reactions of the quinones in the photosynthetic reaction center of Rhodopseudomonas viridis.

The electron-transfer reactions involving the quinones in the bacterial photosynthetic reaction center (bRC) are coupled to a proton uptake by the bRC. In this study, we calculated the energies of the different states of the bRC occurring during these electron-transfer and protonation reactions by an electrostatic model. We considered the possibility that titratable groups of the bRC can change their protonation during these reactions. The protonation probabilities of titratable groups were obtained by a Monte Carlo calculation. In contrast to earlier studies by other groups, we used atomic partial charges derived from quantum-chemical calculations. Our calculated reaction energies are in agreement with experiments. We found that the proton uptake by the bRC is coupled more strongly to changes of the redox state of the quinones than to changes of their protonation state. Thus, the proton uptake by the bRC occurs predominantly before the protonation of QB. According to our computations, the reduction of QB* - to the doubly negative state QB2- is energetically even more unfavorable in the bRC than in solution. Therefore, we suggest that the second electron transfer from QA to QB occurs after QB has received its first proton. We found that the QA. -QB. - state is more populated at pH 7.5 than the QA. -QB.H state. The low population of the QA. -QB.H state may be the reason why the singly protonated QB could not be detected spectroscopically. Our calculations imply that the first protonation of QB. - is a prerequisite for the second electron transfer between QA and QB. Therefore, a pH dependence of the equilibrium between the states QA. -QB. - and QA. -QB. H can also explain the experimentally observed pH dependence of the rate for the second electron-transfer step. On the basis of our calculated reaction energies, we propose the following sequence for the electron-transfer and protonation reactions: (1) first electron transfer from QA to QB, (2) first protonation of QB (at the distal oxygen close to Ser L223), (3) second electron transfer from QA to QB, and (4) second protonation of QB (at the proximal oxygen close to His L190).

Binding Sites↗
PubMed1997

Changes in protonation associated with substrate binding and Cob(I)alamin formation in cobalamin-dependent methionine synthase.

Methionine synthase catalyzes the transfer of a methyl group from methylcobalamin enzyme to homocysteine, generating methionine and cob(I)alamin enzyme, and then from methyltetrahydrofolate to cob(I)alamin enzyme, generating tetrahydrofolate and regenerating the methylcobalamin enzyme. The reactions catalyzed by methionine synthase require deprotonation of the substrate, homocysteine, and protonation of the product tetrahydrofolate, with no net change in proton stoichiometry for a complete turnover cycle. In addition, formation of the intermediate cob(I)alamin enzyme requires a change in the cobalt ligand geometry from 6-coordinate to 4-coordinate, and this rearrangement may require the transient protonation of protein residues to stabilize the cob(I)alamin enzyme. In the E. coli enzyme, the lower face of the methylcobalamin cofactor is coordinated by histidine 759, which is hydrogen bonded to aspartate 757 and then to serine 810, forming a "ligand triad". It has previously been shown that reduction of cob(II)alamin enzyme to cob(I)alamin is associated with the uptake of a proton from solution, and it has been postulated that this proton resides within the His759-Asp757 pair. Cob(I)alamin can also be generated by demethylation of methylcobalamin enzyme by homocysteine; it was not known whether this mode of cob(I)alamin formation was associated with proton uptake. In this paper, we use equilibrium titrations and kinetic analyses in the presence of the pH indicator dye phenol red, along with studies of the pH dependence of oxidation/reduction equilibria, to identify and characterize mechanistic steps associated with proton uptake and release in both the turnover and reactivation of the enzyme. We confirm that cob(I)alamin formation by reduction of cob(II)alamin enzyme is associated with proton uptake and show that mutation of Asp757 to Glu abolishes the pH dependence of this reduction. Demethylation of methylcobalamin enzyme also leads to cob(I)alamin formation and is also shown to be associated with proton uptake. By observing pre-steady-state reactions with homocysteine and methyltetrahydrofolate in the presence of phenol red, we show that this proton uptake occurs at a rate that is equal to the rate of formation of the cob(I)alamin enzyme. In addition, we show that binding of homocysteine to the enzyme results in the rapid release of a proton, presumably the homocysteine thiol proton. In contrast, binding methyltetrahydrofolate to the enzyme does not result in proton uptake, suggesting that the proton destined for the product tetrahydrofolate is already present on the free methylcobalamin enzyme.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗