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 1,189 records · Page 66Linked to original sources
PubMed1978

The microbial metabolism of Cl compounds. The stoicheiometry of respiration-driven proton translocation in Pseudomonas AM1 and in a mutant lacking cytochrome c.

This paper clarifies the role of cytochrome c in Pseudomonas AM1 by measuring the stoicheiometry of proton translocation driven by respiration of endogenous or added substrates in wild-type bacteria and in a mutant lacking cytochrome c (mutant PCT76). The maximum -->H(+)/O ratio (protons translocated out of the bacteria per atom of oxygen consumed during respiration) was about 4 and, except when respiration was markedly affected, this ratio was similar in mutant and wild-type bacteria. The -->H(+)/O ratios were unaltered when the usual oxidase (cytochrome a(3)) was inhibited by 300mum-KCN and respiration involved the single cytochrome b functioning as an alternative oxidase. Ratios measured in cells respiring endogenous substrate and in cells loaded with malate or 3-hydroxybutyrate suggest that there are two proton-translocating segments operating during the oxidation of NADH. By contrast, during oxidation of formaldehyde or methylamine only one pair of protons is translocated. Proton translocation could not be measured with methanol as substrate, because its oxidation was inhibited (90-95%) by 5mm-KSCN. It is tentatively proposed that the electron-transport chain for NADH oxidation in Pseudomonas AM1 is arranged such that the NADH-ubiquinone oxidoreductase forms one proton-translocating segment and the second segment consists of ubiquinone and cytochromes b and a/a(3). The cytochrome c appears to be essential only for respiration and proton translocation from methanol (and possibly from methylamine); there is no conclusive evidence that cytochrome c ever mediates between cytochromes b and a/a(3) in Pseudomonas AM1.

Cyanides↗
PubMed1981

Kinetic solvent isotope effects on the deacylation of specific acyl-papains. Proton inventory studies on the papain-catalysed hydrolyses of specific ester substrates: analysis of possible transition state structures.

1. The hydrolyses of the p-nitrophenyl esters of N-benzyloxycarbonylglycine, alpha-N-benzyloxycarbonyl-L-lysine and N-methoxycarbonyl-L-phenylalanylglycine catalysed by papain (EC 3.4.22.2) have been studied in solvents having a variable composition of 2H2O and H2O. 2. kcat., which represents deacylation in the papain-catalysed hydrolysis of reactive esters, is some 2.3-fold less in 2H2O compared with H2O. The magnitude of kcat. has been determined as a function of the 2H atom fraction of the solvent. 3. Both linear and non-linear methods of least-square regression analysis have been applied to the data in order to obtain best-fit parameter values for several three-parameter models which express kcat. in terms of the 2H atom fraction of the solvent. These models represent some possible modes of restructuring of the active site protonic configuration consequent upon transition state formation. 4. The results of curve fitting reveal an essentially linear dependence of kcat. upon the 2H atom fraction, and it may therefore be concluded that the isotope effect originates from a single proton which is in the process of transfer in the transition state. 5. It is postulated on the basis of this and other evidence that the mobile proton is transferred from an attacking water molecule to the imidazole side chain of His-159 during tetrahedral intermediate formation. This has the effect of stabilizing the transition state and promoting catalysis. The role of His-159 in deacylation is therefore to provide general base catalysis. 6. Models that involve two or more protons, such as a two-proton relay system analogous to that proposed for the serine proteinases, or a multiproton 'medium' effect, are considered unlikely on the basis of the data reported in this paper. 7. A more detailed examination of possible transition state structures reveals that the only structure compatible with available experimental data and consistent with certain theoretical predictions is one in which the proton translocated in concern with reorganization of the heavy atom framework. In addition, the transition state vibrations of the mobile proton are strongly coupled to those of the heavy atoms. These properties of the transition state are also manifest in the transition state for the deacylation of serine proteinases.

Binding Sites↗
PubMed2003

Proton conductance and fatty acyl composition of liver mitochondria correlates with body mass in birds.

The proton conductance of isolated liver mitochondria correlates significantly with body mass in mammals, but not in ectotherms. To establish whether the correlation in mammals is general for endotherms or mammal-specific, we measured proton conductance in mitochondria from birds, the other main group of endotherms, using birds varying in mass over a wide range (nearly 3000-fold), from 13 g zebra finches to 35 kg emus. Respiratory control ratios were higher in mitochondria from larger birds. Mitochondrial proton conductance in liver mitochondria from birds correlated strongly with body mass [respiration rate per mg of protein driving proton leak at 170 mV being 44.7 times (body mass in g)(-0.19)], thus suggesting a general relationship between body mass and proton conductance in endotherms. Mitochondria from larger birds had the same or perhaps greater surface area per mg of protein than mitochondria from smaller birds. Hence, the lower proton conductance was caused not by surface area changes but by some change in the properties of the inner membrane. Liver mitochondria from larger birds had phospholipid fatty acyl chains that were less polyunsaturated and more monounsaturated when compared with those from smaller birds. Phospholipid fatty acyl polyunsaturation correlated positively and monounsaturation correlated negatively with proton conductance. These correlations echo those seen in mammalian liver mitochondria, suggesting that they too are general for endotherms.

Animals↗
PubMed2005

Expression of gp91phox/Nox2 in COS-7 cells: cellular localization of the protein and the detection of outward proton currents.

We have reported previously that gp91phox, expressed in CHO (Chinese hamster ovary) cells, functions as a voltage-dependent proton channel. However, others have reported that COS-7 cells expressing gp91phox failed to exhibit outward proton currents, and concluded that gp91phox does not function as a proton channel. To investigate this clear difference in findings, we have examined the expression and cellular localization of the fusion protein EGFP-C-91, in which gp91phox is fused to the C-terminus of enhanced green fluorescent protein. EGFP-C-91 was observed in the plasma membrane and intracellular membranes of 30% of the transfected COS-7 cells. In the remaining COS-7 cells, EGFP-C-91 was detected in the intracellular membranes only. In CHO cells EGFP-C-91 was present in both the plasma membrane and the intracellular membranes of all transfected cells. Under the whole-cell configuration, outward currents were recorded from COS-7 cells expressing gp91phox. These increased in magnitude and lost their 'droop' over time as the pipette solution equilibrated with the cell cytoplasm (50 min). The threshold activation voltage for the currents was shifted by approximately 60 mV for a 1 unit difference in bath pH. Zn2+ inhibited the outward currents observed in COS-7 cells expressing gp91phox. The tail current reversal potential was -64 mV at a pH(o) (external pH) of 8.0, -40 mV at pH(o) 7.4 and -8 mV at pH(o) 7.0, indicating that the current arises from the movement of protons. Outward currents were exhibited by 37.5% of the COS-7 cells expressing gp91phox. Proton currents were recorded following the excision of inside-out patches from cells transfected with gp91phox. The presence of outward proton currents in COS-7 cells expressing gp91phox provides further support for our proposed role for gp91phox as the NADPH oxidase-associated proton channel.

Animals↗
PubMed1998

Trends in prescribing H2-receptor antagonists and proton pump inhibitors in primary care.

BACKGROUND: H2-receptor antagonists and proton pump inhibitors account for approximately 15% of primary care prescribing costs in the UK. AIM: To examine the use of antisecretory drugs in primary care between October 1991 and September 1996. METHOD: Analysis of prescribing data from an ongoing postal survey performed every 3 months on a rolling quota of 250 UK general practitioners (GPs), identified from a representative sampling frame of 1000 GPs. RESULTS: There were 8811 new courses of proton pump inhibitors and 11,948 new courses of H2-receptor antagonists during this study. The number of new prescriptions for proton pump inhibitors increased by 174.5%, but decreased for H2-receptor antagonists by 12.5%. Proton pump inhibitors were mostly prescribed for reflux disease (52.7%) and H2-receptor antagonists for non-specific dyspepsia (43.6%). Proton pump inhibitors (14.1%) were less likely to be stopped than H2-receptor antagonists (35.3%) overall, and they were less likely to be stopped because of perceived ineffectiveness (5.3%) than H2-receptor antagonists (23.8%). The rate of stopping treatment because of side-effects was about 3% for both classes of drug. CONCLUSIONS: Prescribing of proton pump inhibitors has increased sharply each year since 1991. One reason may be that GPs perceive proton pump inhibitors to be more effective than H2-receptor antagonists.

Anti-Ulcer Agents↗
PubMed2001

Proton pump inhibitors versus H2-antagonists: a meta-analysis of their efficacy in treating bleeding peptic ulcer.

PURPOSE: To evaluate whether proton pump inhibitors are more effective than H2-antagonists (H2-A) for the treatment of bleeding peptic ulcer. DATA SOURCES: PubMed database until January 2000. STUDY SELECTION: Comparative randomized trials of proton pump inhibitors (omeprazole, lansoprazole, or pantoprazole) vs. H2-A (cimetidine, ranitidine or famotidine). DATA EXTRACTION: Meta-analysis combining the odds ratios (OR) of the individual studies in a global OR (Peto method). OUTCOMES EVALUATED: Persistent or recurrent bleeding, need for surgery, or mortality. DATA SYNTHESIS: Eleven studies fulfilled the inclusion criteria and contained data for at least one of the planned comparisons. Persistent or recurrent bleeding was reported in 6.7% (95% CI: 4.9-8.6%) of the patients treated with proton pump inhibitors, and in 13.4% (95% CI: 10.8-16%) of those treated with H2-A (OR 0.4; 95% CI: 0.27-0.59) (chi2-homogeneity test, 18; P=0.09). Surgery was needed in 5.2% (95% CI: 3.4-6.9%) of the patients treated with proton pump inhibitors, and in 6.9% (95% CI: 4.9-8.9%) of the patients treated with H2-A (OR 0.7; 95% CI: 0.43-1.13). Respective percentages for mortality were 1.6% (95% CI: 0.9-2.9%) and 2.2% (95% CI: 1.3-3.7%) (OR 0.69; 95% CI: 0.31-1.57). SUB-ANALYSIS: Five studies evaluated the effect of both therapies given in bolus injections on persistent or recurrent bleeding rate, which was 6% (95% CI: 3.6-8.3%) and 8.1% (95% CI: 5.3-10.9%), respectively (OR, 0.57; 95% CI: 0.31-1.05). Persistent or recurrent bleeding in high risk patients (Forrest Ia, Ib and IIa) occurred in 13.2% (95% CI: 7.9-8%) of the patients treated with proton pump inhibitors and in 34.5% (27-42%) of those treated with H2-A (OR 0.28; 95% CI: 0.16-0.48). In patients not having endoscopic therapy, persistent or recurrent bleeding was reported, respectively, in 4.3% (95% CI: 2.7-6.7%) and in 12% (95% CI: 8.7-15%) (OR 0.24; 95% CI: 0.13-0.43). Less marked differences were observed in patients having adjunct endoscopic therapy: 10.3% (95% CI: 6.7-13.8%) and 15.2% (11.1-19.3%) (OR 0.59; 95% CI: 0.36-0.97). Moreover, the significance disappeared in this group when a single outlier study was excluded. CONCLUSIONS: Proton pump inhibitors are more effective than H2-A in preventing persistent or recurrent bleeding from peptic ulcer, although this advantage seems to be more evident in patients not having adjunct sclerosis therapy. This beneficial effect seems to be similar or even more marked in patients with Forrest Ia, Ib or IIa ulcers. However, proton pump inhibitors are not more effective than H2-A for reducing surgery or mortality rates. Nevertheless, the data are too scarce and heterogeneous to draw definitive conclusions, and further comparative trials are clearly warranted.

Anti-Ulcer Agents↗
PubMed2001

Bedtime H2 blockers improve nocturnal gastric acid control in GERD patients on proton pump inhibitors.

AIM: Proton pump inhibitors taken twice daily before meals (proton pump inhibitor b.d. AC) effectively controls daytime gastric pH; however, nocturnal gastric acid breakthrough (NAB) occurs in more than 75% of patients. Adding an H2-blocker at bedtime decreases NAB in normal subjects. The efficacy of this regimen has not been evaluated in GERD patients. The aim of this study was to assess the effects of proton pump inhibitor b.d., both with and without bedtime H2-blocker on intragastric pH and the occurrence of NAB in GERD patients. METHODS: Prolonged ambulatory pH studies in GERD patients were reviewed. Group A: 60 patients (mean age 53 years, male 30) taking either omeprazole 20 mg or lansoprazole 30 mg b.d. Group B: 45 patients (mean age 49 years, male 23) on proton pump inhibitor b.d. (omeprazole 20 mg or lansoprazole 30 mg) plus an H2-blocker at bedtime (ranitidine 300 mg, famotidine 40 mg or nizatidine 300 mg). Eleven patients were evaluated during treatment with both regimens (group C). The percentage time of nocturnal and daytime intragastric pH > 4 and per cent of patients with gastric NAB were analysed. In the patients with NAB, its duration and associated oesophageal acid exposure also were analysed. RESULTS: Median percentage time intragastric pH > 4 overnight was 51% in group A, compared to 96% in group B (P < 0.0001). Median percentage daytime pH > 4 was 73% in group A and 79.8% in group B (P=0.14). Median percentage time intragastric pH >p 4 overnight increased from 54.6% without H2RA to 96.5% after adding bedtime H2RA (P=0.0013) in group C patients. NAB occurred in 82% patients in group A and 40% in group B (P < 0.0001). The mean duration of oesophageal acid exposure during NAB was significantly shorter in group B (18 +/- 6 min) than in group A (42 +/- 9 min, P=0.04). SUMMARY: Adding a bedtime H2-blocker to the treatment enhanced nocturnal gastric pH control and decreased NAB compared to the proton pump inhibitor b.d. regimen. A bedtime H2-blocker also decreased oesophageal acid exposure during NAB. CONCLUSION: Adding a bedtime H2-blocker to a proton pump inhibitor b.d. regimen should be considered in patients who require continued nocturnal gastric acid control whilst taking proton pump inhibitor b.d.

Anti-Ulcer Agents↗
PubMed2003

Meta-analysis: proton pump inhibitor or H2-receptor antagonist for Helicobacter pylori eradication.

AIM: To compare H2-receptor antagonists and proton pump inhibitors as adjuvants to triple therapy for Helicobacter pylori eradication. METHODS: H. pylori-infected patients with peptic ulcer were randomized to receive either 300 mg nizatidine or 30 mg lansoprazole plus 1 g amoxicillin and 500 mg clarithromycin taken b.d. for 7 days. H. pylori eradication was assessed 4 weeks after therapy. Using meta-analytical techniques, we combined the results of this study with other randomized controlled comparisons of H2-receptor antagonists and proton pump inhibitors as adjuvants to triple therapy. RESULTS: One hundred and one patients were randomized. H. pylori eradication was 94% (47/50) [95% confidence interval (CI), 83-99%] (intention-to-treat) in the H2-receptor antagonist group vs. 86% (44/51) (95% CI, 74-94%) in the proton pump inhibitor group (P = 0.3). There has been a total of 12 similar studies (1415 patients). The overall efficacy was similar in intention-to-treat analysis: 78% (549/701) with H2-receptor antagonists vs. 81% (575/714) with proton pump inhibitors (odds ratio, 0.86; 95% CI, 0.66-1.12). A non-significant trend favouring H2-receptor antagonist (79% vs. 69%; odds ratio, 1.14; 95% CI, 0.76-1.71; P = 0.5) was seen in the comparison of clarithromycin-containing regimens. In contrast, in non-clarithromycin-containing trials, there was a slight, but significant, advantage with proton pump inhibitors (85% vs. 78%; odds ratio, 0.64; 95% CI, 0.45-0.92; P = 0.02). CONCLUSION: Overall, proton pump inhibitor and H2-receptor antagonist antisecretory agents appear to be similarly effective as adjuvants for H. pylori triple therapy. It is unlikely that the direct anti-H. pylori effect of proton pump inhibitors is responsible for their ability to enhance anti-H. pylori therapy.

2-Pyridinylmethylsulfinylbenzimidazoles↗
PubMed2004

Double proton transfer behavior and one-electron oxidation effect in double H-bonded glycinamide-formic acid complex.

The behavior of double proton transfer occurring in a representative glycinamide-formic acid complex has been investigated at the B3LYP/6-311 + + G( * *) level of theory. Thermodynamic and, especially, kinetic parameters, such as tautomeric energy, equilibrium constant, and barrier heights have been discussed, respectively. The relevant quantities involved in the double proton transfer process, such as geometrical changes, interaction energies, and intrinsic reaction coordinate calculations have also been studied. Computational results show that the participation of a formic acid molecule favors the proceeding of the proton transfer for glycinamide compared with that without mediate-assisted case. The double proton transfer process proceeds with a concerted mechanism rather than a stepwise one since no ion-pair complexes have been located during the proton transfer process. The calculated barrier heights are 11.48 and 0.85 kcal/mol for the forward and reverse directions, respectively. However, both of them have been reduced by 2.95 and 2.61 kcal/mol to 8.53 and -1.76 kcal/mol if further inclusion of zero-point vibrational energy corrections, where the negative barrier height implies that the reverse reaction should proceed with barrierless spontaneously, analogous to that occurring between glycinamide and formamide. Furthermore, solvent effects on the thermodynamic and kinetic processes have also been predicted qualitatively employing the isodensity surface polarized continuum model within the framework of the self-consistent reaction field theory. Additionally, the oxidation process for the double H-bonded glycinamide-formic acid complex has also been investigated. Contrary to that neutral form possessing a pair of two parallel intermolecular H bonds, only a single H bond with a comparable strength has been found in its ionized form. The vertical and adiabatic ionization potentials for the neutral complex have been determined to be about 9.40 and 8.69 eV, respectively, where ionization is mainly localized on the glycinamide fragment. Like that ionized glycinamide-formamide complex, the proton transfer in the ionized complex is characterized by a single-well potential, implying that the proton initially attached to amide N4 in the glycinamide fragment cannot be transferred to carbonyl O13 in the formic acid fragment at the geometry of the optimized complex.

Electrons↗
PubMed2005

Activation and protonation of dinitrogen at the FeMo cofactor of nitrogenase.

The protonation of N2 bound to the active center of nitrogenase has been investigated using state-of-the-art density-functional theory calculations. Dinitrogen in the bridging mode is activated by forming two bonds to Fe sites, which results in a reduction of the energy for the first hydrogen transfer by 123 kJ/mol. The axial binding mode with open sulfur bridge is less reactive by 30 kJ/mol and the energetic ordering of the axial and bridged binding modes is reversed in favor of the bridging dinitrogen during the first protonation. Protonation of the central ligand is thermodynamically favorable but kinetically hindered. If the central ligand is protonated, the proton is transferred to dinitrogen following the second protonation. Protonation of dinitrogen at the Mo site does not lead to low-energy intermediates.

Binding Sites↗
PubMed2004

Vertebrate ultraviolet visual pigments: protonation of the retinylidene Schiff base and a counterion switch during photoactivation.

For visual pigments, a covalent bond between the ligand (11-cis-retinal) and receptor (opsin) is crucial to spectral tuning and photoactivation. All photoreceptors have retinal bound via a Schiff base (SB) linkage, but only UV-sensitive cone pigments have this moiety unprotonated in the dark. We investigated the dynamics of mouse UV (MUV) photoactivation, focusing on SB protonation and the functional role of a highly conserved acidic residue (E108) in the third transmembrane helix. On illumination, wild-type MUV undergoes a series of conformational changes, batho --> lumi --> meta I, finally forming the active intermediate meta II. During the dark reactions, the SB becomes protonated transiently. In contrast, the MUV-E108Q mutant formed significantly less batho that did not decay through a protonated lumi. Rather, a transition to meta I occurred above approximately 240 K, with a remarkable red shift (lambda(max) approximately 520 nm) accompanying SB protonation. The MUV-E108Q meta I --> meta II transition appeared normal but the MUV-E108Q meta II decay to opsin and free retinal was dramatically delayed, resulting in increased transducin activation. These results suggest that there are two proton donors during the activation of UV pigments, the primary counterion E108 necessary for protonation of the SB during lumi formation and a second one necessary for protonation of meta I. Inactivation of meta II in SWS1 cone pigments is regulated by the primary counterion. Computational studies suggest that UV pigments adopt a switch to a more distant counterion, E176, during the lumi to meta I transition. The findings with MUV are in close analogy to rhodopsin and provides further support for the importance of the counterion switch in the photoactivation of both rod and cone visual pigments.

Amino Acid Sequence↗
PubMed1974

Proton magnetic resonance spectra of Rhodospirillum rubrum cytochrome c2.

Nuclear magnetic resonance spectra of ferro- and ferricytochrome C(2) from the facultative photoheterotroph, Rhodospirillum rubrum, obtained with a 220-MHz spectrometer, are presented. Assignments to protons of various important structural groups in contact with or near the prosthetic heme group are given. These include (a) in the ferro-form, methyl, cbeta and cgamma protons of the extraplanar ligand residue, methionine 91, and methyl protons of the residue leucine 32; and (b) corresponding protons in the ferri-form with the addition of methyl protons from the condensation of a heme vinyl group with cysteine 17, a single proton from the condensation involving cysteine 14, and protons from the ring methyls. These resonances are compared with those corresponding to the same groups in horse heart cytochrome c.

Chemical Phenomena↗
PubMed1993

Light-driven proton or chloride pumping by halorhodopsin.

Halorhodopsin from Halobacterium halobium was purified and reconstituted with lipids from purple membranes. The resulting protein-containing membrane sheets were adsorbed to a planar lipid membrane and photoelectric properties were analyzed. Depending on light conditions, halorhodopsin acted either as a light-driven chloride pump or as a proton pump: green light caused chloride transport and additional blue light induced proton pumping. In the living cell, both to these vectorial processes would be directed toward the cytoplasm and, compared to ion transport by bacteriorhodopsin, this is an inversed proton flow. Azide, a catalyst for reversible deprotonation of halorhodopsin, enhanced proton transport, and the deprotonated Schiff base in the 13-cis configuration (H410) was identified as the key intermediate of this alternative catalytic cycle in halorhodopsin. While chloride transport in halorhodopsin is mediated by a one-photon process, proton transport requires the absorption of two photons: one photon for formation of H410 and release of a proton, and one photon for photoisomerization of H410 and re-formation of H578 with concomitant uptake of a proton by the Schiff base.

Azides↗
PubMed1996

Kinetic coupling between electron and proton transfer in cytochrome c oxidase: simultaneous measurements of conductance and absorbance changes.

Bovine heart cytochrome c oxidase is an electron-current driven proton pump. To investigate the mechanism by which this pump operates it is important to study individual electron- and proton-transfer reactions in the enzyme, and key reactions in which they are kinetically and thermodynamically coupled. In this work, we have simultaneously measured absorbance changes associated with electron-transfer reactions and conductance changes associated with protonation reactions following pulsed illumination of the photolabile complex of partly reduced bovine cytochrome c oxidase and carbon monoxide. Following CO dissociation, several kinetic phases in the absorbance changes were observed with time constants ranging from approximately 3 microseconds to several milliseconds, reflecting internal electron-transfer reactions within the enzyme. The data show that the rate of one of these electron-transfer reactions, from cytochrome a3 to a on a millisecond time scale, is controlled by a proton-transfer reaction. These results are discussed in terms of a model in which cytochrome a3 interacts electrostatically with a protonatable group, L, in the vicinity of the binuclear center, in equilibrium with the bulk through a proton-conducting pathway, which determines the rate of proton transfer (and indirectly also of electron transfer). The interaction energy of cytochrome a3 with L was determined independently from the pH dependence of the extent of the millisecond-electron transfer and the number of protons released, as determined from the conductance measurements. The magnitude of the interaction energy, 70 meV (1 eV = 1.602 x 10(-19) J), is consistent with a distance of 5-10 A between cytochrome a3 and L. Based on the recently determined high-resolution x-ray structures of bovine and a bacterial cytochrome c oxidase, possible candidates for L and a physiological role for L are discussed.

Animals↗
PubMed2002

The proton channel of the energy-transducing nicotinamide nucleotide transhydrogenase of Escherichia coli.

The nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple direct hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III to proton translocation by the membrane-intercalated domain II. To delineate the proton channel of the enzyme, 25 conserved and semiconserved prototropic amino acid residues of domain II of the Escherichia coli transhydrogenase were mutated, and the mutant enzymes were assayed for transhydrogenation from NADPH to an NAD analogue and for the coupled outward proton translocation. The results confirmed the previous findings of others and ourselves on the essential roles of three amino acid residues and identified another essential residue. Three of these amino acids, His-91, Ser-139, and Asn-222, occur in three separate membrane-spanning alpha helices of domain II of the beta subunit of the enzyme. Another residue, Asp-213, is probably located in a cytosolic-side loop that connects to the alpha helix bearing Asn-222. It is proposed that the three helices bearing His-91, Ser-139, and Asn-222 come together, possibly with another highly conserved alpha helix to form a four-helix bundle proton channel and that Asp-213 serves to conduct protons between the channel and domain III where NADPH binding energy is used via protein conformation change to initiate outward proton translocation.

Amino Acid Sequence↗
PubMed2003

Molecular determinants of proton modulation of glycine receptors.

Extracellular pH regulates glycine receptors through an unknown mechanism. Here we demonstrate that acidic pH remarkably inhibited glycine-activated whole-cell currents in recombinant glycine alpha1 and alpha1beta receptors transiently expressed in human embryonic kidney 293 cells. The proton effect was voltage-independent and pharmacologically competed with glycine receptor agonist glycine and antagonist strychnine. Using site-directed mutagenesis, we have identified an N-terminal domain that is essential for proton-induced inhibition of glycine current. In alpha1 homomers, removal of the hydroxyl group by mutation of residue Thr-112 to Ala or Phe abolished inhibition of glycine currents by acidification. In contrast, mutation of Thr-112 to another hydroxylated residue (Tyr) produced receptors that retained partial proton sensitivity. In alpha1beta heteromers, a single mutation of the beta subunit T135A, which is homologous to alpha1 Thr-112, reduced proton sensitivity, whereas the double mutation alpha1(T112A)beta(T135A) almost completely eliminated the proton sensitivity. In addition, the mutation alpha1 H109A greatly reduced sensitivity to protons in homomeric alpha1 receptors. The results demonstrate that extracellular pH can regulate the function of glycine alpha1 and alpha1beta receptors. An extracellular domain consisting of Thr-112 and His-109 at the alpha1 subunit and Thr-135 at the beta subunit plays a critical role in determining proton modulation of glycine receptor function.

Amino Acid Sequence↗
PubMed2004

The Escherichia coli NADH:ubiquinone oxidoreductase (complex I) is a primary proton pump but may be capable of secondary sodium antiport.

The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. Recently, it was demonstrated that complex I from Klebsiella pneumoniae translocates sodium ions instead of protons. Experimental evidence suggested that complex I from the close relative Escherichia coli works as a primary sodium pump as well. However, data obtained with whole cells showed the presence of an NADH-induced electrochemical proton gradient. In addition, Fourier transform IR spectroscopy demonstrated that the redox reaction of the E. coli complex I is coupled to a protonation of amino acids. To resolve this contradiction we measured the properties of isolated E. coli complex I reconstituted in phospholipids. We found that the NADH:ubiquinone oxidoreductase activity did not depend on the sodium concentration. The redox reaction of the complex in proteoliposomes caused a membrane potential due to an electrochemical proton gradient as measured with fluorescent probes. The signals were sensitive to the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), the inhibitors piericidin A, dicyclohexylcarbodi-imide (DCCD), and amiloride derivatives, but were insensitive to the sodium ionophore ETH-157. Furthermore, monensin acting as a Na(+)/H(+) exchanger prevented the generation of a proton gradient. Thus, our data demonstrated that the E. coli complex I is a primary electrogenic proton pump. However, the magnitude of the pH gradient depended on the sodium concentration. The capability of complex I for secondary Na(+)/H(+) antiport is discussed.

Amino Acids↗
PubMed2004

Five residues in the HtrI transducer membrane-proximal domain close the cytoplasmic proton-conducting channel of sensory rhodopsin I.

Transducer-free sensory rhodopsins carry out light-driven proton transport in Halobacterium salinarum membranes. Transducer binding converts the proton pumps to signal-relay devices in which the transport is inhibited. In sensory rhodopsin I (SRI) binding of its cognate transducer HtrI inhibits transport by closing a cytoplasmic proton-conducting channel necessary for proton uptake during the SRI photochemical reaction cycle. To investigate the channel closure, a series of HtrI mutants truncated in the membrane-proximal cytoplasmic portion of an SRI-HtrI fusion were constructed and expressed in H. salinarum membranes. We found that binding of the membrane-embedded portion of HtrI is insufficient for channel closure, whereas cytoplasmic extension of the second HtrI transmembrane helix by 13 residues blocks proton conduction through the channel as well as full-length HtrI. Specifically the closure activity is localized in this 13-residue membrane-proximal cytoplasmic domain to the 5 final residues, each of which incrementally contributes to reduction of proton conductivity. Moreover, these same residues in the dark incrementally and proportionally increase the pKa of the Asp-76 counterion to the protonated Schiff base chromophore in the membrane-embedded photoactive site. We conclude that this critical region of HtrI alters the dark conformation of SRI as well as light-induced channel opening. The 5 residues in HtrI correspond in position to 5 residues demonstrated on the homologous NpHtrII to interact with the E-F loop of its cognate receptor NpSRII in the accompanying article (Yang, C.-S., Sineshchekov, O., Spudich, E. N., and Spudich, J. L. (2004) J. Biol. Chem. 279, 42970-42976). These results strongly suggest that the membrane-proximal region of Htr proteins interact with their cognate sensory rhodopsin cytoplasmic domains as part of the signal-relay coupling between the proteins.

Archaeal Proteins↗