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PubMed2001

Inhibition of trans-membrane hexacyanoferrate III reductase activity and proton secretion of maize (Zea mays L.) roots by thenoyltrifluoroacetone.

Intact plants can reduce external oxidants by an appearingly trans-membrane electron transport. In vivo an increase in net medium acidification accompanies the reduction of the apoplastic substrate. Up to now, several NAD(P)H dehydrogenases, b-type cytochromes, and a phylloquinone have been identified and partially purified from plant plasma membranes. The occurrence of a quinone in the plasma membrane of maize roots supports the hypothetical model of a proton-transferring redox system, i.e., an electron transport chain with a quinone as mobile electron and proton carrier. In the present study the trans-membrane electron transport system of intact maize (Zea mays L.) roots was investigated. Flow-through and ionostat systems have been used to estimate the electron and proton transport activity of this material. Application of 4,4,4-trifluoro-1-(2-thienyl)-butane-1,3-dione (thenoyltrifluoroacetone) inhibited the reduction of ferricyanide in the incubation solution of intact maize roots up to 70%. This inhibition could not be washed off by rinsing the roots with fresh incubation medium. The acidification of the medium induced after ferricyanide application was inhibited to about 62%. The effects of thenoyltrifluoroacetone on proton fluxes in the absence of ferricyanide have been characterized in a pH-stat system. The net medium acidification by maize roots was inhibited up to 75% by thenoyltrifluoroacetone in the absence of ferricyanide, while dicumarol inhibited net acidification completely. The inhibition of H(+)-ATPase activity was estimated with plasma membrane vesicles isolated by phase partitioning and treated with 0.05% (w/v) Brij 58. ATP-dependent proton gradients and Pi release were measured after preincubation with the effectors. The proton pumping activity by those plasma membrane vesicles was inhibited by dicumarol (53.6%) and thenoyltrifluoroacctone (77.8%), while the release of Pi was unaffected by both inhibitors.

Cell Membrane↗
PubMed1990

Proton dissociation dynamics in the aqueous layer of multilamellar phospholipid vesicles.

The water layers interspacing between the phospholipid membranes of a multilamellar vesicle are 3-10 water layers across and their width is adjusted by osmotic pressure (Parsegian, V.A., et al., 1986. Methods Enzymol. 127:400-416). In these thin water layers we dissolved pyranine (8 hydroxypyrene 1,3,6 trisulfonate), a compound which, upon photo excitation, ejects it hydroxy proton with time constant of 100 psec. (Gutman, M. 1986. Methods Enzymol. 127:522-538). In the present study we investigated how the width of the aqueous layer, the density of phosphomoieties on the membrane's surface and the activity of water in the layer affect the capacity of protons to diffuse out from the electrostatic cage of the excited anion before it decays to the ground state. Using a combination of steady-state and subnanosecond time-resolved fluorescence measurements we determined the average number of proton excited-anion recombinations before the proton escapes from the Coulomb cage. The probability of recombination in thin water layer is significantly higher than in bulk. The factor contributing most to enhancement of recombination is the diminished water activity of the thin aqueous layer. The time frame for proton escape from an electrostatic trap as big as a membrane-bound protein is 3 orders of magnitude shorter than turnover time of membrane-bound enzymes. Thus the effects of local forces on proton diffusion, at the time scale of physiological processes, is negligible.

Arylsulfonates↗
PubMed1987

How do protons cross the membrane-solution interface? Kinetic studies on bilayer membranes exposed to the protonophore S-13 (5-chloro-3-tert-butyl-2'-chloro-4' nitrosalicylanilide).

A simple carrier model describes adequately the transport of protons across lipid bilayer membranes by the weak acid S-13. We determined the adsorption coefficients of the anionic, A-, and neutral, HA, forms of the weak acid and the rate constants for the movement of A- and HA across the membrane by equilibrium dialysis, electrophoretic mobility, membrane potential, membrane conductance, and spectrophotometric measurements. These measurements agree with the results of voltage clamp and charge pulse kinetic experiments. We considered three mechanisms by which protons can cross the membrane-solution interface. An anion adsorbed to the interface can be protonated by a H+ ion in the aqueous phase (protolysis), a buffer molecule in the aqueous phase or water molecules (hydrolysis). We demonstrated that the first reaction cannot provide the required flux of protons: the rate at which H+ must combine with the adsorbed anions is greater than the rate at which diffusion-limited reactions occur in the bulk aqueous phase. We also ruled out the possibility that the buffer is the main source of protons: the rate at which buffers must combine with the adsorbed anions is greater than the diffusion-limited rate when we reduced the concentration of polyanionic buffer adjacent to the membrane-solution interface by using membranes with a negative surface charge. A simple analysis demonstrates that a hydrolysis reaction can account for the kinetic data. Experiments at acid pH demonstrate that the transfer of H+ from the membrane to the aqueous phase is limited by the rate at which OH- combines with adsorbed HA and that the diffusion coefficient of OH- in the water adjacent to the bilayer has a value characteristic of bulk water. Our experimental results demonstrate that protons are capable of moving rapidly across the membrane-solution interface, which argues against some mechanisms of local chemiosmosis.

Kinetics↗
PubMed1995

ATPase activity, IF1 content, and proton conductivity of ESMP from control and ischemic slow and fast heart-rate hearts.

Earlier studies by Rouslin and coworkers showed that, during myocardial ischemia in slow heart-rate species which include rabbits and all larger mammals examined including humans, there is an IF1-mediated inhibition of the mitochondrial ATPase due to an increase in the amount of IF1 bound to the ATPase (Rouslin, W., and Pullman, M.E., J. Mol. Cell. Cardiol. 19,661-668, 1987). Earlier work by Guerrieri and colleagues demonstrated that IF1 binding to bovine heart ESMP was accompanied by parallel decreases in ATPase activity and in passive proton conduction (Guerrieri, F., et al., FEBS Lett. 213, 67-72, 1987). In the present study rabbit was used as the slow heart-rate species and rat as the fast heart-rate species. Rat is a fast heart-rate species that contains too little IF1 to down regulate the ATPase activity present. Mitochondria were prepared from control and ischemic hearts and ESMP were made from aliquots by sonication at pH 8.0 with 2 mM EDTA. Oligomycin-sensitive ATPase activity and IF1 content were measured in SMP prepared from the control and ischemic mitochondrial samples. After identical incubation procedures, oligomycin-sensitive ATPase activity, oligomycin-sensitive proton conductivity, and IF1 content were also measured in ESMP samples. The study was undertaken to corroborate further what appear to be fundamental differences in ATPase regulation between slow and fast heart-rate mammalian hearts evident during total myocardial ischemia. Thus, passive proton conductivity was used as an independent measure of these regulatory differences. The results show that, consistent with the low IF1 content of rat heart cardiac muscle mitochondria, control rat heart ESMP exhibit approximately twice as much passive proton conductivity as control rabbit heart ESMP regardless of the pH of the incubation and assay. Moreover, while total ischemia caused an increase in IF1 binding and a commensurate decrease in passive proton conductivity in rabbit heart ESMP regardless of pH, neither IF1 content nor proton conductivity changed significantly in rat heart ESMP as a result of ischemia.

Adenosine Triphosphate↗
PubMed2005

Modelling of proton and metal exchange in the alginate biopolymer.

Acid-base behaviour of a commercial sodium alginate extracted from brown seaweed (Macrocystis pyrifera) has been investigated at different ionic strengths (0.1<or=I/mol l(-1)<or=1.0) and in different supporting electrolytes (Et4NI, NaCl, KCl, LiCl, NaCl+MgCl2), with the aim of examining the influence of ionic medium on the proton-binding capacity and of quantifying the strength of interaction with light metal ions in the perspective of speciation studies in natural aqueous systems. Potentiometric ([H+]-glass electrode) and titration calorimetric data were expressed as a function of the dissociation degree (alpha) using different models (Henderson-Hasselbalch modified, Högfeldt three parameters and linear equations). The dependence on ionic strength of the protonation constants was taken into account by a modified specific interaction theory model. Differences among different media were explained in terms of the interaction between polyanion and metal cations of the supporting electrolytes. Quantitative information on the proton-binding capacity, together with the stabilities of different species formed, is reported. Protonation thermodynamic parameters, at alpha=0.5, are log K H=3.686+/-0.005, DeltaG 0=-21.04+/-0.03 kJ mol(-1), DeltaH 0=4.8+/-0.2 kJ mol(-1) and TDeltaS 0=35.7+/-0.3 kJ mol(-1), at infinite dilution. Protonation enthalpies indicate that the main contribution to proton binding arises from the entropy term. A strict correlation between DeltaG and TDeltaS was found, TDeltaS=-9.5-1.73 DeltaG. Results are reported in light of building up a chemical complexation model of general validity to explain the binding ability of naturally occurring polycarboxylate polymers and biopolymers. Speciation profiles of alginate in the presence of sodium and magnesium ions, naturally occurring cations in natural waters, are also reported.

Alginates↗
PubMed2006

Is the risk for secondary cancers after proton therapy enhanced distal to the Planning Target Volume? A two-case report with possible explanations.

It is often assumed that radiation-induced secondary cancer after proton therapy forms preferentially close to the distal fall-off of the spread-out Bragg peak because of an increased relative biological effectiveness (RBE) with regard to cancer induction of low-energy protons. In this study we analyze to what extent dose gradients distal to the Planning Target Volume (PTV) may, independently from the RBE, contribute to enhanced radiation carcinogenesis. The study is based on two dogs which, out of 30 dogs treated with proton therapy at the Paul Scherrer Institute (PSI), developed a secondary cancer. Both dogs were originally diagnosed and treated for a fibrosarcoma and developed an osteosarcoma 48 and almost 60 months, respectively, after radiotherapy. From the dose distributions of the initial radiotherapy for both dogs three-dimensional maps of secondary cancer complication probability (SCCP) were computed. The SCCP maps were analyzed in the regions where the dogs developed a secondary cancer. The SCCP maps showed an enhanced risk in the regions of the femur where the secondary cancers were detected, as compared to the SCCP of the total femur. Excess risk of radiation-induced cancer at the distal part of proton radiation fields can thus be explained using SCCP calculations on the basis of the physical dose distributions. Therefore, the occurrence of secondary cancer close to the distal dose gradients of proton therapy is not necessarily due to an increased RBE of low-energy protons. More extensive studies based on more patients will be necessary to further elucidate the factors influencing the development of secondary tumors.

Animals↗
PubMed1997

Space proton flux and the temporal distribution of cardiovascular deaths.

The influence of solar activity (SA) and geomagnetic activity (GMA) on human homeostasis has long been investigated. The aim of the present study was to analyse the relationship between monthly proton flux (> 90 MeV) and other SA and GMA parameters and between proton flux and temporal (monthly) distribution of total and cardiovascular-related deaths. The data from 180 months (1974-1989) of distribution in the Beilinson Campus of the Rabin Medical Centre, Israel, and of 108 months (1983-1991) from the Kaunas Medical Academy, were analysed and compared with SA, GMA and space proton flux (> 90 MeV). It was concluded: (1) monthly levels of SA, GMA and radiowave propagation (Fof2) are significantly and adversely correlated with monthly space proton flux (> 90 MeV); (2) medical-biological phenomena that increase during periods of low solar and/or geomagnetic activity may be stimulated by physical processes provoked by the concomitant increase in proton flux; (3) the monthly number of deaths related (positively or negatively) to SA are significantly and adversely related to the space proton flux (> 90 MeV).

Adult↗
PubMed2003

Proton conduction through full-length gp91phox requires histidine 115.

The NADPH oxidase of neutrophils is a transmembrane electron transfer complex, containing a flavin adenine dinucleotide and two hemes, all of which are suggested to be contained within gp91 (phox), one of four subunits of the enzyme. The transfer of electrons through the NADPH oxidase is associated with an efflux of protons. gp91 (phox) has previously been demonstrated to function as the proton conduction pathway. The mutation of histidines 111, 115, and 119 to leucines and of histidine 115 to leucine within the N-terminal 230-amino-acid fragment of gp91 (phox) has previously been demonstrated to result in the loss of proton conduction through this N-terminal fragment. In this paper we have investigated the role of these histidines in proton conduction by the full-length gp91 (phox). Stable CHO cell lines were established which expressed full-length gp91 (phox) in which histidines 111, 115, and 119 had been mutated to leucines (CHO91H111/115/119) and in which histidine 115 had been mutated to leucine (CHO91H115L). The expression of gp91 (phox) and its cellular localisation in these cell lines were comparable between wild-type and the mutant gp91 (phox). The mutation of histidines 111, 115, and 119 to leucines or just histidine 115 to leucine resulted in an almost total loss of both the arachidonate-activated influx and efflux of protons, in comparison with that observed for wild-type gp91 (phox). Therefore, histidine 115 is required for proton conduction by both full-length gp91 (phox) and the N-terminal 230-amino-acid fragment of gp91 (phox). Histidine 115 has recently been proposed to act as a coordinating ligand for the outer heme iron of the NADPH oxidase. On the basis of observations for cytochrome c oxidase, we propose a model for this dual role of histidine 115.

Animals↗
PubMed2006

Utilization of methyl proton resonances in cross-saturation measurement for determining the interfaces of large protein-protein complexes.

Cross-saturation experiments allow the identification of the contact residues of large protein complexes (MW>50 K) more rigorously than conventional NMR approaches which involve chemical shift perturbations and hydrogen-deuterium exchange experiments [Takahashi et al. (2000) Nat. Struct. Biol., 7, 220-223]. In the amide proton-based cross-saturation experiment, the combined use of high deuteration levels for non-exchangeable protons of the ligand protein and a solvent with a low concentration of (1)H(2)O greatly enhanced the selectivity of the intermolecular cross-saturation phenomenon. Unfortunately, experimental limitations caused losses in sensitivity. Furthermore, since main chain amide protons are not generally exposed to solvent, the efficiency of the saturation transfer directed to the main chain amide protons is not very high. Here we propose an alternative cross-saturation experiment which utilizes the methyl protons of the side chains of the ligand protein. Owing to the fast internal rotation along the methyl axis, we theoretically and experimentally demonstrated the enhanced efficiency of this approach. The methyl-utilizing cross-saturation experiment has clear advantages in sensitivity and saturation transfer efficiency over the amide proton-based approach.

Computer Simulation↗
PubMed1984

Temperature dependence of light-induced proton movement in reconstituted purple membrane.

Bacteriorhodopsin (BR) was incorporated into phosphatidylcholine (PC) vesicles containing different amounts of other lipids. Under the conditions of nullified membrane potential, light-induced proton movement seemed to follow a kinetic scheme which assumed the existence of a proton-pumping inhibition process characterized by a rate constant, kI. The temperature dependence of both kI and the membrane proton leak rate constant (kD) obeyed a simple Arrhenius equation. The presence of cholesterol in the membrane significantly increased the activation energy (Ea) of both the inhibition and leak process. However, further addition of phosphatidic acid (PA) suppressed the increase of Ea associated with kI. The initial proton pumping rate (R0) of vesicles reconstituted with PC showed a bell-shaped temperature dependence with a maximum at approximately 20 degrees C. The addition of cholesterol abolished this dependence. These results suggest that the molecular origin of the inhibition process characterized by kI is different from that of R0 or kD. The temperature dependence of the steady-state fluorescence polarization of dansylated bacteriorhodopsin in vesicles was also investigated. The polarization of the labels in the vesicles without cholesterol showed a bell-shaped temperature dependence with a maximum at approximately 20 degrees C. However, in the presence of cholesterol, the polarization increased linearly as temperature decreased. A comparison of these results with the observed proton movement in similarly reconstituted systems with unmodified protein indicates that membranes with a low fluidity and negatively charged surfaces enhance proton pumping efficiency of bacteriorhodopsin.

Bacteriorhodopsins↗
PubMed1984

1H NMR of glycosaminoglycans and hyaluronic acid oligosaccharides in aqueous solution: the amide proton environment.

The exchangeable amide protons of hyaluronic acid (HA) oligosaccharides and a higher-molecular-weight segment dissolved in H2O at pH 2.5 or 5.5 were examined by H NMR spectroscopy at 250 MHz. The HA segment preparation showed a single amide resonance, near the chemical shift for the amide proton of the monosaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose (beta-GlcNAc). Smaller HA oligosaccharides showed two or three separate amide proton resonances, corresponding in relative peak area to interior or end GlcNAc residues. The interior GlcNAc amide resonance occurred at the same chemical shift as the single resonance of the HA segment. For the end GlcNAc residues, linkage to D-glucuronopyranose (GlcUA) through C1 resulted in an upfield shift relative to the beta-anomer of GlcNAc, whereas linkage through C3 resulted in a downfield shift relative to the corresponding anomer of GlcNAc. These chemical-shift perturbations appeared to be approximately offsetting in the case of linkage at both positions. The amide proton vicinal coupling constant (ca. 9 Hz) was found to be essentially independent of chain length, residue position, or solution pH. These data favor a nearly perpendicular orientation for the acetamido group with respect to the sugar ring, little affected by linkage of GlcNAc to GlcUA. No evidence for the existence of a stable hydrogen bond linking the amide proton with the carboxyl(ate) oxygen of the adjacent uronic acid residue was found. The amide proton resonances for chondroitin, chondroitin 4-sulfate, and dermatan sulfate were compared to that of HA. The chemical shifts of these resonances deviated no more than 0.1 ppm from that of HA. A small dependence on the identity of the adjacent uronic acid residue was noted, based on the observation of two resonances for dermatan sulfate.

Amides↗
PubMed1986

Respiration-linked proton flux in Wolinella succinogenes during reduction of N-oxides.

Formate uncoupled proton translocation in formate-grown Wolinella succinogenes cells supplied with N-oxides as terminal electron acceptors. In suspensions containing KSCN (but not valinomycin), H2 supported proton translocation when NO3-, NO2-, and NO were provided as oxidants. H+/N-oxide ratios were 4.77 for NO3-, 2.49 for NO2-, and 1.75 for NO. KSCN inhibits N2O reduction thus precluding use of N2O as oxidant. Repeated exposure of cells to NO inhibited their ability to translocated protons with NO as oxidant but only slightly diminished and did not eliminate their capacity for NO3(-)- or NO2(-)-dependent proton flux. Substituting reduced benzyl viologen for H2 and measuring proton uptake provided results consistent with an extramembranal location for the N- oxide reductases. The uncoupler, carbonyl cyanide m-chlorophenylhydrazone, collapsed proton gradients, permitted uptake of 2 mol H+/mol NO3- or NO2-, but unaccountably inhibited NO3- reduction by 50% while leaving H+ uptake stoichiometry of the cells unaffected.

Bacteria↗
PubMed1992

Anomalous effect of uncouplers on respiratory chain-linked transhydrogenation in Escherichia coli membranes: evidence for a localized proton pathway?

Energization of the pyridine nucleotide transhydrogenase in everted membrane vesicles from Escherichia coli JM83 was compared with the process in vesicles of the same strain transformed with the plasmid pDC21 overexpressing this enzyme. Proton translocation was assayed by the quenching of the fluorescence of the probe quinacrine. Agents able to discharge transmembrane proton gradients such as nigericin and the uncouplers 3,3',4',5-tetrachlorosalicylanilide and carbonyl cyanide m-chlorophenylhydrazone inhibited ATP-dependent transhydrogenation of NADP by NADH and discharged transmembrane proton gradients generated by transhydrogenation of AcNAD by NADPH, by oxidation of NADH, and by hydrolysis of ATP. This was observed in everted membrane vesicles of both strains JM83 and JM83pDC21. These strains differed significantly in the response of the NADH oxidation-dependent transhydrogenase. This reaction was inhibited by nigericin and uncouplers in membrane vesicles of JM83 but there was little inhibition or the reaction was stimulated in JM83pDC21, in spite of the discharge of the NADH oxidation-generated proton gradient measured by quinacrine fluorescence in the latter strain. It is proposed that the transhydrogenase is energized by direct or local (nonbulk phase) proton translocation in membranes of this strain. Uncouplers might facilitate these routes but would not discharge them. The generality of these observations was shown using other strains. NADH oxidase activity was severalfold lower in membrane vesicles of JM83pDC21 compared with JM83. The levels of ubiquinone and cytochromes, and the activities of NADH dehydrogenases I and II, and of cytochrome oxidase, were similar in the two strains. It is concluded that the NADH oxidase activity of JM83pDC21 is low because of the reduced rate of collision between electron-transferring complexes of the respiratory chain due to the large amount of transhydrogenase protein in the membranes of this strain. The large amount of transhydrogenase favors direct, nonbulk phase proton transfer. Transhydrogenase activity was stimulated by Ca2+, Mg2+, or Mn2+.

Cell Membrane↗
PubMed1977

Proton evolution from photosystem II. Stoichiometry and mechanistic considerations.

1. In a sequence of flashes given to dark-adapted chloroplasts, the flash yield of proton release oscillates with a period of 4, which is similar but not identical to the oscillation of the O2 flash yield. 2. Using the proton release associated with ferricyanide reduction as a calibration, we computed that two protons are released in the terminal O2-liberating reaction; the other two protons are released in precursor conversion steps. 3. Analysis of the effect of preflashes on the oscillation pattern showed that the S1 leads to S2 transition releases no proton, the S0 leads to S1 transition somewhat less than one (0.75), and the S2 leads to S3 transition somewhat more than one (1.25). 4. The precision of the data was sufficient to exclude the possibility that in the four-step water oxidation, proton release follows a simple 1, 0, 1, 2 pattern. A possible model to interpret the observed flash yield patterns is discussed.

Chemical Phenomena↗
PubMed1987

The role of fixed and mobile buffers in the kinetics of proton movement.

We derive a simple expression for the effective diffusion coefficient of protons in Fick's second law, Deff, when both spatially fixed, HF, and mobile, HM, buffers are present. These buffers are present at moderately high concentrations ([Ftot], [Mtot] greater than 1 mM) in most biological systems. We consider only the case where the protonation reactions remain at equilibrium during the diffusion process. When the pH is to the alkaline side of the pK values of the fixed and mobile buffers ([H+] less than KF, KM), the effective diffusion coefficient of protons in Ficks second law is: (Formula: see text) where DH is the diffusion coefficient of the protons free in the aqueous phase and DHM is the diffusion coefficient of the mobile buffer. The equation illustrates three features of diffusion in a buffered system. Firstly, the effective diffusion coefficient of protons is always lower than the diffusion coefficient of free protons. Secondly, increasing the concentration of fixed buffers always decreases Deff. Thirdly, increasing the concentration of mobile buffer can increase Deff when fixed buffers are present.

Biological Transport↗
PubMed1994

Anomalous response of oxonol-V to membrane potential in mitochondrial proton pumps.

The response of the fluorescent membrane potential probe oxonol-V (bis[3-phenyl 5 oxoisoxazol-4-yl]pentamethine oxonol) in submitochondrial particles (SMP) was dependent upon whether the potential (inside positive) was generated by active proton pumps or by valinomycin-aided passive K+ influx. The fluorescence intensity showed a decrease in the former case and an increase in the latter situation. This anomalous behavior was not observed with other similar anionic probes. Gradual inhibition of proton pumping activity showed that the difference in the response of oxonol-V is not due to possible difference in the magnitude of membrane potential generated in these two situations. In the presence of membrane permeant anions such as TPB- (tetraphenyl boron) or chlorate, the direction of response of oxonol-V fluorescence was the same in both situations. Time-resolved fluorescence of the oxonol-V-SMP system showed three populations: one free form (fluorescence lifetime approximately 60 ps) and two SMP-bound forms (lifetimes of 0.45 ns and 1.4 ns). A fourth population was created during the action of proton pumps. The shorter lifetime (approximately 250 ps) of this new bound form suggest this population to be an aggregated form. This population was absent during the action of proton pumps in the presence of TPB- or chlorate. These results suggest the creation of a charge separated state during the action of proton pumps. The decrease in fluorescence intensity could be the result of aggregation of oxonol-V around the positive end of a proton pump existing in a dipole or charge separated state.

Animals↗
PubMed1994

The light-driven proton pump, cruxrhodopsin-2 in Haloarcula sp. arg-2 (bR+, hR-), and its coupled ATP formation.

Haloarcula sp. arg-2, a natural bacterial isolate from Andes heights, has a light-driven proton pump but not a light-driven anion pump. We have cloned and sequenced the gene encoding for the proton pump which has been named cruxrhodopsin-2. The gene consists of 768 bp encoding 255 amino acids with a molecular mass of 27,544 Da. The deduced amino acid sequence of cruxrhodopsin-2 is 77%, 50%, 48% and 48% identical to those of cruxrhodopsin-1, bacteriorhodopsin, archaerhodopsin-1 and archaerhodopsin-2, respectively. The charged amino acids important for the proton pump function were conserved among all these molecules. Cruxrhodopsin-2 accounted for 0.05 nmol/mg protein in arg-2, which was 20-30-fold less than the proportion of bacteriorhodopsin in Halobacterium salinarium R1M1. In contrast to R1M1, under anaerobic conditions, arg-2 showed light-induced proton extrusion concomitant with an increase in ATP level without transient proton uptake. Dicyclohexylcarbodiimide enhanced the rate and extent of proton extrusion and inhibited ATP formation in the light. The apparent stoichiometry of H+/ATP was estimated to be more than three in this natural bR+hR- strain.

Adenosine Triphosphate↗
PubMed1994

Proton uptake by cytochrome c oxidase on reduction and on ligand binding.

On reduction, cytochrome oxidase was found to take up 2.4 +/- 0.1 protons in the pH range 7.2-8.5, of which 2 are associated with the binuclear centre, and the remaining fractional proton with haem a/CuA. Ligation to oxidised cytochrome oxidase of the azide, formate, fluoride or cyanide anions is accompanied by uptake of one proton. In the case of the reduced enzyme, no protonation changes are observed on binding O2 (Hallén S. and Nilsson T. (1992) Biochemistry 31, 11853-11859) or CO. Cyanide binding to reduced oxidase is, in contrast, still accompanied by uptake of a proton. These findings are discussed in terms of our previously-published proposal for the ligand chemistry of the binuclear site. The results overall suggest a principle of electroneutrality of redox and ligand state changes of the binuclear centre, with charge compensations provided only by protonation reactions.

Animals↗