Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Protons”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 901 records · Page 50Linked to original sources
PubMed2001

Comparative treatment planning between proton and X-ray therapy in pancreatic cancer.

With the utilization of new biologic agents and experimental chemotherapy in the treatment of pancreatic cancer, the issue of local-regional control will become increasingly important. This study was undertaken to determine the feasibility of dose escalation using proton therapy, as compared to conventional 3-dimensional conformal radiation, by minimizing the dose to normal tissues. The photon treatment plans of 4 patients with unresectable pancreatic cancer treated on a biologic therapy trial were utilized. Each patient was treated using a 3- or 4-field photon plan with 45 Gy to the clinical target volume (CTV), followed by a boost of 14.4 Gy to the gross target volume (GTV). Using a Helax treatment planning system, proton plans were generated to encompass the same CTV and GTV to the same prescribed dose. Dose-volume histograms (DVHs) were generated for the GTV, CTV, spinal cord, liver, and right and left kidneys. Each DVH was compared between the photon and proton plans. Proton plans utilized either a 2- or 3-field technique. Available energies included 130 or 180 MeV. Range modulators and bolus were used as needed to conform to the target volume. With the CTV and GTV receiving the same dose from the proton and photon plans, all individual proton plans were superior to the photon plans in reduction of normal tissue dose. For the 4 patients, the average dose reduction to 50% of the organ at risk was 78% to spinal cord (p = 0.003), 73% to left kidney (p = 0.025), 43% to right kidney (p = 0.059), and 55% to liver (p = 0.061). These comparative treatment plans show proton therapy results in significant reductions of dose to normal tissue compared to conventional photons while treating the same target volumes. This allows for the design of dose-escalation protocols using protons in combination with new biologic therapies and chemotherapy.

Adenocarcinoma↗
PubMed1999

Proton-bound cluster ions in ion mobility spectrometry.

Gaseous oxygen and nitrogen bases, both singly and as binary mixtures, have been introduced into ion mobility spectrometers to study the appearance of protonated molecules, and proton-bound dimers and trimers. At ambient temperature it was possible to simultaneously observe, following the introduction of molecule A, comparable intensities of peaks ascribable to the reactant ion (H2O)nH+, the protonated molecule AH+ and AH+ H2O, and the symmetrical proton bound dimer A2H+. Mass spectral identification confirmed the identifications and also showed that the majority of the protonated molecules were hydrated and that the proton-bound dimers were hydrated to a much lesser extent. No significant peaks ascribable to proton-bound trimers were obtained no matter how high the sample concentration. Binary mixtures containing molecules A and B, in some cases gave not only the peaks unique to the individual compounds but also peaks due to asymmetrical proton bound dimers AHB+. Such ions were always present in the spectra of mixtures of oxygen bases but were not observed for several mixtures of oxygen and nitrogen bases. The dimers, which were not observable, notable for their low hydrogen bond strengths, must have decomposed in their passage from the ion source to the detector, i.e. in a time less than approximately 5 ms. When the temperature was lowered to -20 degrees C, trimers, both homogeneous and mixed, were observed with mixtures of alcohols. The importance of hydrogen bond energy, and hence operating temperature, in determining the degree of solvation of the ions that will be observed in an ion mobility spectrometer is stressed. The possibility is discussed that a displacement reaction involving ambient water plays a role in the dissociation.

Air↗
PubMed2002

Coupling of electron and proton transport in photosynthetic membranes: molecular mechanism.

Using the method of Modified Neglect of Diatomic Overlap (MNDO), the electronic structure of plastoquinol (PQH(2)) and plastoquinone (PQ) in neutral, singly (PQ(-)) and doubly (PQ(2-)) reduced states is studied. The conformational analysis performed on these molecules shows that in the lowest energy conformation, the angle between the first link of the tail backbone and the ring plane of neutral and singly reduced PQ and plastoquinol is nearly the same and differs by 15 degrees from that of doubly reduced PQ. Nevertheless, for all states of plastoquinone and for plastoquinol, the total energy changes by less than 0.2 eV when the studied angle is varied from 0 degrees to 180 degrees. As in Rhodobacter sphaeroides, the oxygen of the PQ ring is reported to form a hydrogen bond with a nitrogen in the ring of Histidine (His) L 190. The energy of the PQ-His complex was calculated for different redox states of PQ and for several values of the distance between the molecules (N-O distance from 0.2 to 0.5 nm). For every considered complexes, the total energy dependence on the proton position on the line connecting the N and O atoms was determined, to see if the hydrogen bond is formed. It is shown that for only singly reduced PQ this dependence has a symmetric two-well form, i.e. the hydrogen bond is formed. For neutral and doubly reduced PQ, the curve is also two-well but asymmetric, so that the proton is bound to His or to PQ, correspondingly. On the basis of these results, we propose the following scheme of electron-proton coupling. Negatively charged oxygens of PQ form H-bonds with proton donor groups of the surrounding protein and fix PQ in its pocket. While the negative charges of oxygens increase after quinone reduction, protons shift to PQ oxygens and form strong hydrogen bonds with them. Upon second PQ reduction, protons are torn away from surrounding amino acids and form covalent bonds with the quinol. Resulting PQH(2) detaches from its binding place and is replaced by a neutral PQ. The lacking protons on amino acids in the Q(B) pocket are replaced by a step-by-step transfer from the stroma bulk through the proton channels.

Electrons↗
PubMed1995

Terminal base pairs of oligodeoxynucleotides: imino proton exchange and fraying.

We have estimated the dissociation constant of the terminal base pairs of the B-DNA duplexes formed by 5'-d(CGCGATCGCG) and 5'-d(TAGCGCTA) by two methods, one based on the change in imino proton chemical shift with temperature and the other on the apparent pK shift of the imino proton, as monitored by the change in chemical shift of aromatic protons. These methods do not rely on imino proton exchange, whose rate was also measured. (1) The effect of ammonia on the imino proton exchange rate of the terminal pair of the 5'-d(CGCGATCGCG) duplex is 67 times less than on the isolated nucleoside. This provides an upper limit on the exchange rate from the closed pair. In fact, the effect is just as predicted from the dissociation constant, assuming that there is no exchange at all from the closed pair and that, as has been argued previously, external catalysts act on the open state as they do on the isolated nucleoside. The inhibition of catalyzed proton exchange in the closed pair, despite exposure of one face of the pair to solvent, is a new feature of the exchange process. It will allow determination of the dissociation constant of terminal pairs from the exchange rate. (2) Intrinsic catalysis of proton exchange is less efficient for the terminal pair than for an internal one. A possible explanation is that proton transfer across the water bridge responsible for intrinsic catalysis is slower, as expected if the open-state separation of the bases is larger in a terminal pair. This observation may lead to a direct method for the study of fraying. (3) At 0 degrees C, the dissociation constant of the second pair of the 5'-d(CGCGATCGCG) duplex is close to the square of the constant for the terminal pair, as predicted from a simple model of fraying. The enthalpy and entropy of opening of the terminal pairs may be compared with those of nearest neighbor interactions derived from calorimetry [Breslauer, K. J., et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 3746-3750].

Base Composition↗
PubMed1995

Proton NMR investigation of the [4Fe--4S]1+ cluster environment of nitrogenase iron protein from Azotobacter vinelandii: defining nucleotide-induced conformational changes.

This work presents the complete assignment of the isotropically shifted 1H NMR resonances of Azotobacter vinelandii nitrogenase iron protein (Fe protein) to beta-CH2 and alpha-CH protons of the [4Fe--4S]1+ cluster cysteinyl ligands. Four resonances were observed for the reduced Fe protein with chemical shifts of 49, 23, 17, and 13 ppm. T1 measurements and analysis of relative peak areas coupled with one-dimensional nuclear Overhauser effect (NOE) difference spectra were used to assign the two most downfield-shifted resonances (49 and 23 ppm) to cysteinyl ligand beta-CH2 protons and the 17 and 14 ppm resonances to cysteinyl ligand alpha-CH protons. Temperature dependent studies of the isotropically shifted protons revealed both Curie and anti-Curie behavior. These results, along with previous Mossbauer studies of the Fe protein, allowed the assignment of signal A (49 ppm) to four beta-CH2 protons and signal C (17 ppm) to 2 alpha-CH protons of two cysteinyl ligands bound to a mixed-valence iron pair (Fe3(+)--Fe2+) of the [4Fe--4S]1+ cluster. Signal B (23 ppm) was assigned to four beta-CH2 protons, and signal C (17 ppm) and D (13 ppm) were assigned to two alpha-CH protons of two cysteinyl ligands bound to a ferrous pair of irons (2Fe2+). The effects of MgATP, MgADP, and Mg-adenosine-beta, gamma-methylene-5'-triphosphate binding to the Fe protein on the assigned resonances were established and are discussed in the context of nucleotide-induced changes in the protein environment of the [4Fe--4S] cluster.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1993

Reaction of bulk protons with a mitochondrial inner membrane preparation: time-resolved measurements and their analysis.

The laser-induced proton pulse technique [Gutman, M. (1986) Methods Enzymol. 127, 522-538] was applied on suspensions of submitochondrial vesicles, and the exchange of protons between the bulk and the mitochondrial membranes was measured in the time-resolved domain with a submicrosecond resolution. The protons were discharged by photoexcitation of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) by a short laser pulse, and the reprotonation of the pyranine anion was monitored at 457.8 nm. In parallel, the protonation of the membrane was followed at 496.5 nm, looking at the transient absorbance of fluorescein, covalently attached to the M side of the membrane. The analysis of the relaxation dynamics was carried out by a simulation procedure that reconstructs the observed dynamics of the two chromophores. The analysis revealed the presence of the membrane indigenous buffering moieties. The low-pK buffer (pK 4.1) was present in a quantity of 100 +/- 20 nmol/mg of protein, and its kinetics indicate that it appears in multianionic clusters bearing a negative electric charge. The medium-pK buffer (pK 6.9) was present in a larger quantity (200 +/- 20 nmol/mg), and its kinetic parameter indicated clustering into positively charged domains. Both types of indigenous buffer reacted with the proton and pyranine anion in unhindered diffusion-controlled reactions. On the other hand, the exchange of protons between the indigenous buffer moieties was rather slow. No evidence was found for the presence of sites capable of retaining a proton, secluded from the bulk, for a time frame longer than 100 microseconds as required by the models of localized proton gradient.

Anions↗
PubMed1991

NMR structural studies of intramolecular (Y+)n.(R+)n(Y-)nDNA triplexes in solution: imino and amino proton and nitrogen markers of G.TA base triple formation.

We reported previously on NMR studies of (Y+)n.(R+)n(Y-)n DNA triple helices containing one oligopurine strand (R)n and two oligopyrimidine strands (Y)n stabilized by T.AT and C+.GC base triples [de los Santos, C., Rosen, M., & Patel, D. J. (1989) Biochemistry 28, 7282-7289]. Recently, it has been established that guanosine can recognize a thymidine.adenosine base pair to form a G.TA triple in an otherwise (Y+)n.(R+)n(Y-)n triple-helix motif. [Griffin, L. C., & Dervan, P. B. (1989) Science 245, 967-971]. The present study extends the NMR research to the characterization of structural features of a 31-mer deoxyoligonucleotide that folds intramolecularly into a 7-mer (Y+)n.(R+)n(Y-)n triplex with the strands linked through two T5 loops and that contains a central G.TA triple flanked by T.AT triples. The G.TA triplex exhibits an unusually well resolved and narrow imino and amino exchangeable proton and nonexchangeable proton spectrum in H2O solution, pH 4.85, at 5 degrees C. We have assigned the imino protons of thymidine and amino protons of adenosine involved in Watson-Crick and Hoogsteen pairing in T.AT triples, as well as the guanosine imino and cytidine amino protons involved in Watson-Crick pairing and the protonated cytidine imino and amino protons involved in Hoogsteen pairing in C+.GC triples in the NOESY spectrum of the G.TA triplex. The NMR data are consistent with the proposed pairing alignment for the G.TA triple where the guanosine in an anti orientation pairs through a single hydrogen bond from one of its 2-amino protons to the 4-carbonyl group of thymidine in the Watson-Crick TA pair.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Composition↗
PubMed1991

Resonance Raman study on intact pea phytochrome and its model compounds: evidence for proton migration during the phototransformation.

Resonance Raman (RR) scattering from intact pea phytochrome was observed in resonance with the blue band at ambient temperature. The relative populations of the red-light-absorbing form (Pr) and far-red-light-absorbing form (Pfr) under laser illumination were estimated from the absorption spectra. The most prominent RR band of Pr obtained by 364-nm excitation under 740-nm pumping exhibited a frequency shift between H2O and D2O solutions, but that of Pfr obtained by 407-nm excitation under 633-nm pumping did not, indicating a distinct difference in a protonation state of their chromophores. Since the protonation level of a whole molecule of intact phytochrome remains unchanged between Pr and Pfr, this observation indicates migration of a proton from the chromophore of Pr to the protein moiety of Pfr. As model compounds, octaethylbiliverdin (OEBV-h3), its deuterated and 15N derivatives, and their protonated forms were also studied with both RR and 1H and 15N NMR spectroscopies. The RR spectrum of the protonated form, for which the protonation site was determined to be C-ring pyrrole nitrogen by NMR, displayed a deuteration shift corresponding to that of Pr, suggesting a similar protonated structure for the pyrrolic rings of Pr. The RR spectral difference between OEBV-h3 and OEBV-d3 and that between H2O and D2O solutions of Pfr suggested that the N-H protons of the A-, B-, and D-rings of intact phytochrome are replaced with deuterons in D2O. A role of the 7-kDa segment of phytochrome is discussed on the basis of RR spectral differences between the intact and large phytochromes.

Biliverdine↗
PubMed2001

Effect of metal binding on electrogenic proton transfer associated with reduction of the secondary electron acceptor (QB) in Rhodobacter sphaeroides chromatophores.

The influence of metal ion (Cd(2+), Zn(2+), Ni(2+)) binding on the electrogenic phases of proton transfer connected with reduction of quinone Q(B) in chromatophores from Rhodobacter sphaeroides was studied by time-resolved electric potential changes. In the presence of metals, the electrogenic transients associated with proton transfer on first and second flash at pH 8 were found to be slower by factors of 3-6. This is essentially the same effect of metal binding that was observed on optical transients in isolated reaction centers (RC), where the metal ion was shown to inhibit proton transfer [Paddock, M. L., Graige, M. S., Feher, G., and Okamura, M. Y. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, 6183-6188]. The effect of metal binding on the kinetics in chromatophores is, therefore, similarly attributed to inhibition of proton uptake, which becomes rate-limiting. A striking observation was an increase in the amplitude of the electrogenic proton-uptake phase after the first flash with bound metal ion. We attribute this to a loss of internal proton rearrangement, requiring that the protons that stabilize Q(B)(-) come from solution. In mutant RCs, in which His-H126 and His-H128 are replaced with Ala, the apparent binding of Cd(2+) and Ni(2+) was decreased, showing that the binding site of these metal ions is the same as found in RC crystals [Axelrod, H. L., Abresch, E. C., Paddock, M. L., Okamura, M. Y., and Feher, G. (2000) Proc. Natl. Acad. Sci. U.S.A. 97, 1542-1547]. Therefore, the unique proton entry point near His-H126, His-H128, and Asp-M17 that was identified in isolated RCs is also the entry point in chromatophores.

Alanine↗
PubMed1992

Proton transfer during the reaction between fully reduced cytochrome c oxidase and dioxygen: pH and deuterium isotope effects.

The pH dependence of proton uptake and electron transfers during the reaction between fully reduced cytochrome c oxidase and oxygen has been studied using the flow-flash method. Proton uptake was monitored using different pH indicators. We have also investigated the effect of D2O on the electron-transfer reactions. Proton uptake was biphasic throughout the pH range studied (6.3-9.3), and the decrease of the observed rate constants at increasing pH could be described by titration curves with pKa values of 8-8.5. Of the four phases resolved in the redox reaction, the rate constants for the first two were independent of pH, whereas that of the third decreased at increasing pH with a pKa of 7.9. All phases except the first were slower in D2O than in H2O. The values obtained for kH/kD were 1.0 for the first phase, 1.4 for the second and third phases, and 2.5 for the fourth phase. We suggest from these results that the fast phase of proton uptake is initiated by the second phase of the redox reaction and that this step includes a partially rate-limiting internal proton transfer. The third and fourth phases of the redox reaction are suggested to be rate limited by proton uptake from the medium. The pH dependencies of the proton uptake reactions are consistent with the participation of a titrable group in the protein in proton transfer from the medium to the oxygen-binding site.

Animals↗
PubMed1994

The dynamics of proton transfer at the C side of the mitochondrial membrane: picosecond and microsecond measurements.

The excited-state proton emitter, pyranine (8-hydroxypyrene-1,3,6-trisulfonate), was introduced into the inner aqueous space of inside-out submitochondrial particles (SMP). Upon initiation of respiration, the dye recorded acidification of this space. Incorporation of high concentrations of the dye (approximately 100 nmol/mg of protein) had no effect on the respiratory functions of the vesicles, nor on their capacity to execute delta microH(+)-coupled reverse electron transfer. The respiratory control ratio (RCR) remained as high as RCR > 4. Pulse irradiation of the dye caused photodissociation of the proton from the 8-hydroxy position. The release of the proton and its reaction with the matrix of the inner space of SMP were monitored at two time intervals: nanosecond fluorimetry measured the dissociation of the proton from the excited dye molecule (phi OH*), while microsecond spectroscopy followed the reaction between the proton and the ground-state anion (phi O-). Numerical integration of the differential rate equations describes the diffusion of protons in the perturbed system. The nanosecond measurements yield the physical characteristics of the aqueous phase that dissolves the dye. The apparent dielectric constant of that space is rather low (epsilon = 20). The diffusion coefficient of the proton is 2.3 x 10(-5) cm2/s, and the activity of water is aH2O = 0.87. All of these values imply that a large fraction of the intervesicular aqueous phase is taken up by the hydration layer of the lipids and proteins of the C side of the membrane. The microsecond dynamics measurements indicate that the rates of proton binding to the membrane surface components reach an equilibrium within 60 microseconds.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Enoyl-coenzyme A hydratase-catalyzed exchange of the alpha-protons of coenzyme A thiol esters: a model for an enolized intermediate in the enzyme-catalyzed elimination?

3-Quinuclidinone catalyzes the exchange of the alpha-protons of butyryl-coenzyme A (CoA) with a second-order rate constant of 2.4 x 10(-6) M-1 s-1. In contrast, enoyl-CoA hydratase catalyzes the stereospecific exchange of the pro-2S proton of butyryl-CoA with a maximum second-order rate constant of ca. 8 x 10(2) M-1 s-1. This isotope exchange reaction is completely stereospecific within the limits of experimental detection (over 600-fold). The enzyme-catalyzed exchange is dependent on pD, decreasing above a pKa of 8.8 and below a pKa of 8.1, but independent of the buffer concentration. The stereospecificity of the exchange was unexpected because the pro-2R hydrogen is abstracted during the enzyme-catalyzed dehydration of 3(S)-hydroxybutyryl-CoA. In spite of the ability to exchange the pro-2S hydrogen, the stereospecificity of the dehydration reaction was determined to be better than 1 in 10(5) as no incorporation of 2H into the alpha-position of crotonyl-CoA or into the pro-2S position of 3(S)-hydroxybutyryl-CoA was detected during prolonged equilibrations with enoyl-CoA hydratase. Both the exchange of the alpha-proton and the dehydration activity of the enzyme are diminished by over 100-fold in a site-directed mutation of rat liver enoyl-CoA hydratase, where glutamate-164 is changed to glutamine, strongly suggesting that the same active site base is responsible for proton abstraction in both the dehydration and solvent exchange reactions. The enoyl-CoA hydratase-catalyzed exchange of the alpha-protons becomes nonstereospecific when the acidity of the alpha-protons is enhanced. While alpha-proton abstraction can be observed when no elimination reaction is possible, there is no evidence for proton abstraction without elimination in the crotonase equilibrations with 3(S)-hydroxybutyryl-CoA, 3-hydroxypropionyl-CoA, or 3-chloropropionyl-CoA. The differences in the isotope exchange and dehydration reactions emphasize the importance of the 3-hydroxyl group in promoting elimination and are consistent with a concerted elimination mechanism.

Acyl Coenzyme A↗
PubMed1982

Proteolipid of adenosinetriphosphatase from yeast mitochondria forms proton-selective channels in planar lipid bilayers.

Proteolipid isolated from yeast mitochondrial adenosinetriphosphatase by butanol extraction is reincorporated into lipid vesicles from which planar membranes are formed. The proteolipid permits electric conductance through the membrane. This conductance occurs through membrane channels which are highly selective for protons. Proton channels in the membrane are directly observed at high proton concentrations in the aqueous phases. Channels open and close independently from each other; their open-state conductances and lifetimes are monodisperse but influenced by the applied voltage (12 pS and 3 s, respectively, at pH 2.2 and 100 mV). Proton channels do not occur in single proteolipid molecules; the conducting structure consists of at least two polypeptide chains since channels form in a (reversible) bimolecular reaction of nonconducting forms of proteolipid. The number of proton channels at a constant proteolipid concentration changes in sharp transitions and by orders of magnitudes upon critical changes of membrane composition and pH. These transitions are caused by transitions of proteolipid organization in the membrane from a dispersed state (equilibrium between channel-forming "dimers" and a large pool of "monomers") to a state of almost complete aggregation of proteolipid which stabilizes large proton-conducting structures (probably associates of channel-forming dimers). This self-association of isolated proteolipid into structures containing proton-selective channels suggests that the six proteolipids in the adenosinetriphosphatase complex exist as a self-associating entity containing most likely three proton channels.

Adenosine Triphosphatases↗
PubMed1989

Dynamics of proton diffusion within the hydration layer of phospholipid membrane.

The diffusion of protons at the immediate vicinity of (less than 10 A from) a phospholipid membrane is studied by the application of the laser-induced proton pulse. A light-sensitive proton emitter (8-hydroxypyrene-1,3,6-trisulfonate) was trapped exclusively in the hydration layers of multilamellar vesicles made of egg phosphatidylcholine, and the protons were dissociated by a synchronizing laser pulse. The recombination of the proton with pyranin anion was monitored by time-resolved spectroscopy and analyzed by a diffusion-controlled formalism. The measured diffusion coefficient is only slightly smaller than the diffusion coefficient of proton in bulk water. Modulating the width of the hydration layer by external pressure had a direct effect on the diffusibility of the proton: the narrower the hydration layer, the slower is the diffusion of protons.

Arylsulfonates↗
PubMed2003

FTIR spectroscopic characterization of the cytochrome aa3 from Acidianus ambivalens: evidence for the involvement of acidic residues in redox coupled proton translocation.

The aa(3)-type quinol oxidase from Acidianus ambivalens is a divergent member of the heme-copper oxidases superfamily, namely, concerning the putative channels for intraprotein proton conduction. In this study, we used electrochemically induced FTIR difference spectroscopy to identify residues involved in redox-coupled protonation changes. In the spectral region characteristic for the nu(C=O) mode from protonated aspartic or glutamic acid side chains, a number of prominent features can be observed between 1790 and 1710 cm(-)(1), clearly indicating the reorganization or protonation of more than four protonatable residues upon electron transfer. A direct comparison of the Fourier-transform infrared difference spectra at different pH values reveals the noteworthy high pK of >8 for some of these residues, and the protonation of two of them. These acidic residues may play a role in the proton transport to the oxygen reducing site, in proton pumping pathways, or in protonation reactions concomitant with quinone reduction. Whereas the residues contributing between 1790 and 1750 cm(-)(1) have the typical position of an aspartic/glutamic acid side chain buried in the protein, a position closer to the surface is suggested for the residues contributing below approximately 1730 cm(-)(1). The possible involvement of residues contributing between 1750 and 1720 cm(-)(1) in the quinone binding site is demonstrated on the basis of experiments in the presence and absence of ubiquinone-2 and of the native electron carrier of the A. ambivalens respiratory chain, caldariella quinone. Most signals seen here are not observable in comparable spectra of typical members of the heme copper oxidase superfamily and thus reflect unique features of the enzyme from the hyperthermoacidophilic archaeon A. ambivalens.

Binding Sites↗
PubMed2003

Proton transport by proteorhodopsin requires that the retinal Schiff base counterion Asp-97 be anionic.

At pH >7, proteorhodopsin functions as an outward-directed proton pump in cell membranes, and Asp-97 and Glu-108, the homologues of the Asp-85 and Asp-96 in bacteriorhodopsin, are the proton acceptor and donor to the retinal Schiff base, respectively. It was reported, however [Friedrich, T. et al. (2002) J. Mol. Biol., 321, 821-838], that proteorhodopsin transports protons also at pH <7 where Asp-97 is protonated and in the direction reverse from that at higher pH. To explore the roles of Asp-97 and Glu-108 in the proposed pumping with variable vectoriality, we compared the photocycles of D97N and E108Q mutants, and the effects of azide on the photocycle of the E108Q mutant, at low and high pH. Unlike at high pH, at a pH low enough to protonate Asp-97 neither the mutations nor the effects of azide revealed evidence for the participation of the acidic residues in proton transfer, and as in the photocycle of the wild-type protein, no intermediate with unprotonated Schiff base accumulated. In view of these findings, and the doubts raised by absence of charge transfer after flash excitation at low pH, we revisited the question whether transport occurs at all under these conditions. In both oriented membrane fragments and liposomes reconstituted with proteorhodopsin, we found transport at high pH but not at low pH. Instead, proton transport activity followed the titration curve for Asp-97, with an apparent pK(a) of 7.1, and became zero at the pH where Asp-97 is fully protonated.

Aspartic Acid↗
PubMed2003

Mechanisms of redox-coupled proton transfer in proteins: role of the proximal proline in reactions of the [3Fe-4S] cluster in Azotobacter vinelandii ferredoxin I.

The 7Fe ferredoxin from Azotobacter vinelandii (AvFdI) contains a [3Fe-4S](+/0) cluster that binds a single proton in its reduced level. Although the cluster is buried, and therefore inaccessible to solvent, proton transfer from solvent to the cluster is fast. The kinetics and energetics of the coupled electron-proton transfer reaction at the cluster have been analyzed in detail by protein-film voltammetry, to reveal that proton transfer is mediated by the mobile carboxylate of an adjacent surface residue, aspartate-15, the pK of which is sensitive to the charge on the cluster. This paper examines the role of a nearby proline residue, proline-50, in proton transfer and its coupling to electron transfer. In the P50A and P50G mutants, a water molecule has entered the cluster binding region; it is hydrogen bonded to the backbone amide of residue-50 and to the Asp-15 carboxylate, and it is approximately 4 A from the closest sulfur atom of the cluster. Despite the water molecule linking the cluster more directly to the solvent, proton transfer is not accelerated. A detailed analysis reveals that Asp-15 remains a central part of the mechanism. However, the electrostatic coupling between cluster and carboxylate is almost completely quenched, so that cluster reduction no longer induces such a favorable shift in the carboxylate pK, and protonation of the base no longer induces a significant shift in the pK of the cluster. The electrostatic coupling is crucial for maintaining the efficiency of proton transfer both to and from the cluster, over a range of pH values.

Amino Acid Substitution↗
PubMed2005

Strongly hydrogen-bonded water molecule present near the retinal chromophore of Leptosphaeria rhodopsin, the bacteriorhodopsin-like proton pump from a eukaryote.

Leptosphaeria rhodopsin (LR) is an archaeal-type rhodopsin found in fungi, and is the first light-driven proton-pumping retinal protein from eukaryotes. LR pumps protons in a manner similar to that of bacteriorhodopsin (BR), a light-driven proton pump of haloarchaea. The amino acid sequence of LR is more homologous to that of Neurospora rhodopsin (NR) than BR, whereas NR has no proton-pumping activity. These facts raise the question of how the proton-pumping function is achieved. In this paper, we studied structural changes of LR following the retinal photoisomerization by means of low-temperature Fourier transform infrared (FTIR) spectroscopy, and compared the obtained spectra with those for BR and NR. While the light-induced photoisomerization from the all-trans to 13-cis form was commonly observed among LR, BR, and NR, we found that the structural changes of LR are closer to those of BR than to those of NR in terms of detailed vibrational bands of retinal and protein. The most prominent difference was seen for the water O-D stretching vibrations (measured in D2O). LR exhibits an O-D stretch of water at 2257 cm(-1), indicating the presence of a strongly hydrogen-bonded water molecule. Such strongly hydrogen-bonded water molecules (O-D stretch at <2400 cm(-1)) were observed for BR, but not for NR. Comprehensive studies of BR mutants and archaeal rhodopsins have revealed that strongly hydrogen-bonded water molecules are found only in the proteins exhibiting proton-pumping activity, suggesting that strongly hydrogen-bonded water molecules and transient weakening of their binding are essential for the proton-pumping function of rhodopsins. This observation for LR provided additional experimental evidence of the correlation between strongly hydrogen-bonded water molecules and proton-pumping activity of archaeal rhodopsins.

Amino Acid Sequence↗