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PubMed1991

On the nature of the mitochondrial proton leak.

Respiring mitochondria have a significant passive permeability to protons; the mechanism of this proton leak is unknown. Several putative mechanisms were tested. Mitochondrial permeability to small sugars was unaffected by energization, suggesting that there is no significant dielectric breakdown at high membrane potential. Mitochondria are argued to have a proton permeability that is 6 to 8 orders of magnitude higher than the permeability to other cations, suggesting that the proton leak is probably not via a simple pore or membrane defect. 15-30% of the proton leak of freshly prepared mitochondria was extractable with bovine serum albumin and is probably due to fatty acids. Little if any of the proton leak appears to be due to cycling of ions other than protons, or to be associated with the functional activity of the proton pumps. The mitochondrial proton leak shares several properties with the proton permeability of pure phospholipid bilayers, suggesting that they share the same mechanism, although the leak through the bilayer in mitochondria may be modified by the presence of proteins.

Animals↗
PubMed2002

Proteorhodopsin is a light-driven proton pump with variable vectoriality.

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions. The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.

Animals↗
PubMed2002

Proton bound homodimers and heterodimers of amides and amines in the gas phase. Equilibrium studies by Fourier transform ion cyclotron resonance spectrometry.

By injection of the proton bound homodimer [DMF.H+.DMF] of N,N-dimethylformamide (DMF) generated in an external ion source into a mixture of DMF and a second base within the cell of a Fourier transform ion cyclotron resonance (FT-ICR) spectrometer the equilibria between [DMF.H+.DMF] and the other possible proton bound dimers [DMF.H+.base] and [base.H+.base] have been studied for 13 different bases. Strongly polar bases like aliphatic amides and dimethyl sulfoxide (DMSO) exchange both DMF in [DMF.H+.DMF] by a two step process, while the almost non-polar amines exchange only one DMF. If the base is a primary or secondary amine, the proton bound heterodimer [DMF.H+.amine] reacts further by the addition of one DMF to create a proton bound trimer [(DMF)2.H+.amine]. The affinity deltaG(DMFH+) of the bases towards protonated DMF relative to neutral DMF depends linearly on the difference deltaGB of the gas phase basicity of DMF and the other base, but different correlation lines are obtained for polar and non-polar ligands (deltaGDMFH+ = 0.44GB(base)-375 [kJ/mol] (r = 0.97) and deltaGDMFH+ = 0.46GB(base)-397 [kJ/mol] (r = 0.99), respectively). This different behavior is explained by a different character of the proton bridge in the heterodimers containing only polar ligands and those incorporating a non-polar ligand besides DMF. The former dimers contain a more or less symmetric proton bridge while the latter can be viewed as a protonated base solvated by DMF. The available data have been used to calculate the molecular pair gas phase basicity of DMF and the 13 bases used and to estimate the dissociation energies of the bonds of the proton bridge in various proton bound heterodimers.

Algorithms↗
PubMed2000

Incidence of secondary Helicobacter pylori resistance to antibiotics in treatment failures after 1-week proton pump inhibitor-based triple therapies: a prospective study.

BACKGROUND: Antibiotic-resistant Helicobacter pylori strains are becoming increasingly prevalent, although it is not clear to what extent the new resistant organisms will spread. AIM: To evaluate the incidence of secondary Helicobacter pylori resistance to metronidazole, clarithromycin and/or amoxycillin after one-week proton pump inhibitor based triple therapy failure in patients who were, before therapy infected with Helicobacter pylori strains susceptible to these antibiotics. PATIENTS AND METHODS: Enrolled in the study were 97 consecutive Helicobacter pylori-positive subjects infected by Helicobacter pylori strains susceptible to metronidazole, clarithromycin and amoxycillin. Activity of tested antibiotics was determined by means of the E-test. Patients were treated for seven days with a proton pump inhibitor, omeprazole 20 mg twice daily or pantoprazole 40 mg once daily, plus clarithromycin 250 mg twice daily and metronidazole 250 mg four times daily; or with a proton pump inhibitor plus amoxycillin 1 g twice daily and clarithromycin 500 mg twice daily. Two months after completion of therapy, endoscopy and gastric biopsies for histology, rapid urease test and culture were repeated. RESULTS: Four patients were dropped from the study Overall Helicobacter pylori cure rates expressed as both intention-to-treat and per-protocol analyses, were, respectively 80% (40/50) and 81.6% (40/49) with proton pump inhibitor, clarithromycin and metronidazole and 76.6% (36/47) and 81.8% (36/44) with proton pump inhibitor amoxycillin and clarithromycin. No significant differences were observed between the two treatments. Subjects in whom treatment failed were significantly younger and had less active ulcer than cured patients. Of treatment failures, 70.6% (12 out of 17 subjects) de veloped a secondary resistance to metronidazole (35.33% and/or clarithromycin (64.7%). Secondary antibiotic resistance occurred in 77. 8% of treatment failures treated with proton pump inhibitor, clarithromycin and metronidazole and in 62.5% of those treated with proton pump inhibitor, amoxycillin and clarithromycin. Considering all patients treated, the overall incidence of secondary metronidazole and/or clarithromycin resistance after therapy was reported in 12.9% of subjects (12 out of 93 treated patients). CONCLUSIONS: Secondary Helicobacter pylori resistances to metronidazole and/or clarithromycin occurred in large percentages in patients with treatment failure after the one-week proton pump inhibitor-based triple therapies, proton pump inhibitor, clarithromycin and metronidazole and proton pump inhibitor, amoxycillin and clarithromycin. It is likely that new antibiotics or treatment strategies will be needed in the near future to successfully treat Helicobacter pylori infection.

Adult↗
PubMed2004

A mechanistic investigation of the enhanced cleavage at histidine in the gas-phase dissociation of protonated peptides.

Enhanced gas-phase cleavage of peptides adjacent to histidine was investigated. The peptides examined were angiotensins III (RVYIHPF) and IV (VYIHPF) as well as synthetic peptide analogues with altered key residues ((R)VYI-X-Z-F; X = F or H and Z = A, P, or Sar) or a fixed charge M3P(+)CH(2)C(O)-VYIHPF. While all singly protonated peptide ions containing both histidine and arginine fragment nonselectively, the doubly protonated peptide ions with arginine and histidine, and the singly protonated peptides containing histidine but not arginine, cleave in a selective manner. In particular, dominant complementary b+/y+ product ions resulting from cleavage between the HP amide bond are observed. For the fixed-charge derivative, selective cleavage occurs only if a proton is added to produce a doubly charged precursor. The results are consistent with involvement of a protonated histidine in the selective cleavage. The ratio of b+/y+ is determined by the identity of the residue C-terminal to histidine and by the ability of protonated histidine to transfer a proton to the C-terminal leaving fragment. This was probed further by systematically changing the residue C-terminal to histidine and by alkylating histidine. The results indicate that while b+/y+ complementary ion pairs dominate in doubly protonated RVYIHPF, b5(2+) and b6(2+) product ions dominate the spectra of doubly protonated RVYIHAF. Also, dominant b5(2+) product ions are observed when the histidine side chain is alkylated (H) in doubly protonated RVYIHPF. Based on all of the results, a selective fragmentation mechanism for enhanced cleavage at histidine involving an atypical b ion structure is proposed.

Amino Acid Sequence↗
PubMed2001

Dynamics of the proton transfer reaction on the cytoplasmic surface of bacteriorhodopsin.

The cytoplasmic surface of bacteriorhodopsin is characterized by a group of carboxylates that function as a proton attractive domain [Checover, S., Nachliel, E., Dencher, N. A., and Gutman, M. (1997) Biochemistry 36, 13919-13928]. To identify these carboxylates, we selectively mutated them into cysteine residues and monitored the effects of the dynamics of proton transfer between the bulk and the surface of the protein. The measurements were carried out without attachment of a pH-sensor to the cysteine residue, thus avoiding any structural perturbation and change in the surface charge caused by the attachment of a reporter group, and the protein was in its BR state. The purple membranes were suspended in an unbuffered solution of pyranine (8-hydroxypyrene-1,3,6-trisulfonate) and exposed to a train of 1000 laser pulses (2.1 mJ/pulse, lambda = 355 nm, at 10 Hz). The excitation of the dye ejected the hydroxyl's proton, and a few nanoseconds later, a pair of free protons and ground-state pyranine anion was formed. The experimental observation was the dynamics of the relaxation of the system to the prepulse state. The observed signals were reconstructed by a numeric method that replicates the chemical reactions proceeding in the perturbed space. The detailed reconstruction of the measured signal assigned the various proton-binding sites with rate constants for proton binding and proton exchange and the pK values. Comparison of the results obtained by the various mutants indicates that the dominant proton-binding cluster of the wild-type protein consists of D104, E161, and E234. The replacement of D104 or E161 with cysteine lowered the proton binding capacity of the cluster to approximately 60% of that of the native protein. The replacement of E234 with cysteine disrupted the structure of the cluster, causing the two remaining carboxylates to function as isolated residues that do not interact with each other. The possibility of proton transfer between monomers is discussed.

Anions↗
PubMed1982

Kinetics for exchange of imino protons in the d(C-G-C-G-A-A-T-T-C-G-C-G) double helix and in two similar helices that contain a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and an extra adenine, d(C-G-C-A-G-A-A-T-T-C-G-C-G).

The relaxation lifetimes of imino protons from individual base pairs were measured in (I) a perfect helix, d(C-G-C-G-A-A-T-T-C-G-C-G), (II) this helix with a G . C base pair replaced with a G . T base pair, d(C-G-T-G-A-A-T-T-C-G-C-G), and (III) the perfect helix with an extra adenine base in a mismatch, d(C-G-C-A-G-A-A-T-T-C-G-C-G). The lifetimes were measured by saturation recovery proton nuclear magnetic resonance experiments performed on the imino protons of these duplexes. The measured lifetimes of the imino protons were shown to correspond to chemical exchange lifetimes at higher temperatures and spin-lattice relaxation times at lower temperatures. Comparison of the lifetimes in these duplexes showed that the destabilizing effect of the G . T base pair in II affected the opening rate of only the nearest-neighbor base pairs. For helix III, the extra adenine affected the opening rates of all the base pairs in the helix and thus was a larger perturbation for opening of the base pairs than the G . T base pair. The temperature dependence of the exchange rates of the imino proton in the perfect helix gives values of 14-15 kcal/mol for activation energies of A . T imino protons. These relaxation rates were shown to correspond to exchange involving individual base pair opening in this helix, which means that one base-paired imino proton can exchange independent of the others. For the other two helices that contain perturbations, much larger activation energies for exchange of the imino protons were found, indicating that a cooperative transition involving exchange of at least several base pairs was the exchange mechanism of the imino protons. The effects of a perturbation in a helix on the exchange rates and the mechanisms for exchange of imino protons from oligonucleotide helices are discussed.

Adenine↗
PubMed1987

Aconitase: its source of catalytic protons.

An ordinary isotope partition experiment was performed to determine the rate of dissociation of the proton from the donor site for the hydration of cis-aconitate. Aconitase in [3H]water was efficiently diluted into well-mixed solutions of cis-aconitate. Citrate and isocitrate that were formed within 2 s were more heavily labeled than could be explained by consideration of an isotope effect in the processing of one proton per enzyme equivalent. Control experiments indicate that mixing was much more rapid than catalytic turnover, ruling out incompletely diluted [3H]water as a significant isotope source. Therefore, it appears that significantly more than one enzyme-bound tritium atom (protons) must have been used in the course of the multiple turnover of the enzyme after the dilution was complete. Isotope incorporation reached values in excess of four proton equivalents as a limit with simple Michaelis dependence on cis-aconitate. From the half-saturation concentration value for trapping, 0.15 mM, the t 1/2 for exchange of each of these protons with solvent appears to be approximately 0.1 s at 0 degrees C. The large number of protons trapped seems to suggest the existence of a structurally stabilized pool of protons, or water, that communicates between the active site base and the medium in the hydration of cis-aconitate. The proton abstracted in the dehydration of [3H]citrate is transferred directly to undissociated cis-aconitate to form isocitrate without dilution, or cis-aconitate having dissociated, the tritium passes to the medium, presumably through the pool of bound protons indicated above. All of the citrate-derived protons can be found in isocitrate if cis-aconitate is added in sufficient concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Aconitate Hydratase↗
PubMed1990

Processes of base-pair opening and proton exchange in Z-DNA.

Using proton magnetic resonance, we have investigated imino and amino proton exchange in the Z form of the four oligomers d(Cbr8GCGCbr8G), d(CGm5CGCG), d(CG)6, and d(CG)12. In the latter two oligomers, all five exchangeable protons have been assigned. We find that proton acceptors such as NH3 or the basic form of Tris enhance imino proton exchange. The base-pair lifetime can then be obtained by extrapolation of the exchange time to infinite concentration of proton acceptor. For d(CG)6 and d(CG)12, the values are ca. 3.5 ms at 80 degrees C and ca. 130 ms at 35 degrees C. The latter value is about 65 times longer than in the same oligomers in the B form. The activation energy of base-pair opening, 80 kJ/mol, is the same in the Z and the B forms of d(CG)12. At 5 degrees C, the base-pair lifetime is about 3 s, much smaller than the time constant of the Z to B transition, to which it is therefore unrelated. The base-pair dissociation constant at 35 degrees C, 0.5 X 10(-6), is 5 times smaller than for the same oligomers in the B form. In the absence of added catalyst, at pH 7, the exchange time of the imino proton is 30 min at 5 degrees C. That of both cytidine amino protons, assigned by NOE, is about 50 min. The longest proton exchange time, ca. 330 min, is assigned unambiguously to the guanosine amino protons. Thus assigned and interpreted in terms of exchange chemistry rather than structural kinetics, the exchange times do not support earlier models of Z-DNA internal motions.

Base Composition↗
PubMed2001

Proton-collecting properties of bovine heart cytochrome C oxidase: kinetic and electrostatic analysis.

Proton-transfer reactions on the surface of bovine heart cytochrome c oxidase were investigated by combining a laser-induced proton-pulse technique with molecular modeling. The experimental approach simultaneously monitors the state of pyranine protonation in the bulk phase and that of a fluorescein indicator specifically attached to the native Cys(III-115) residue of subunit III of cytochrome oxidase. The reversible dynamics of the acid-base equilibration between the surface and the bulk phase were measured with submicrosecond time resolution and analyzed by numerical integration of coupled nonlinear differential rate equations. Kinetic analysis shows that carboxylates on the surface of the protein act as a proton-collecting antenna, which is able to rapidly transfer protons to nearby histidines that function as a local proton reservoir. These properties enable cytochrome oxidase to carry out its redox-linked proton translocation. Molecular modeling of the fluorescein-binding site indicates that, in addition to the covalent bond, the dye is anchored through a hydrogen bond to the hydroxyl moiety of Tyr(VII-50). The protonation of the dye is mediated through three residues that shuttle protons between the bulk and the dye. A correlation between the measured kinetic properties of the bound fluorescein and the different configurations of the dye allows us to predict the identity of the proton-binding sites in the fluorescein-binding domain.

Animals↗
PubMed2005

The conserved histidine 295 does not contribute to proton cotransport by the glutamate transporter EAAC1.

Transmembrane glutamate transport by the excitatory amino acid carrier (EAAC1) is coupled to the cotransport of three Na(+) ions and one proton. Previously, we suggested that the mechanism of H(+) cotransport involves protonation of the conserved glutamate residue E373. However, it was also speculated that the cotransported proton is shared in a H(+)-binding network, possibly involving the conserved histidine 295 in the sixth transmembrane domain of EAAC1. Here, we used site-directed mutagenesis together with pre-steady-state electrophysiological analysis of the mutant transporters to test the protonation state of H295 and to determine its involvement in proton transport by EAAC1. Our results show that replacement of H295 with glutamine, an amino acid residue that cannot be protonated, generates a fully functional transporter with transport kinetics that are close to those of the wild-type EAAC1. In contrast, replacement with lysine results in a transporter in which substrate binding and translocation are dramatically inhibited. Furthermore, it is demonstrated that the effect of the histidine 295 to lysine mutation on the glutamate affinity is caused by its positive charge, since wild-type-like affinity can be restored by changing the extracellular pH to 10.0, thus partially deprotonating H295K. Together, these results suggest that histidine 295 is not protonated in EAAC1 at physiological pH and, thus, does not contribute to H(+) cotransport. This conclusion is supported by data from H295C-E373C double mutant transporters which demonstrate that these residues cannot be linked by oxidation, indicating that H295 and E373 are not close in space and do not form a proton binding network. A kinetic scheme is used to quantify the results, which includes binding of the cotransported proton to E373 and binding of a modulatory, nontransported proton to the amino acid side chain in position 295.

Amino Acid Substitution↗
PubMed2005

G204D, a mutation that blocks the proton-conducting D-channel of the aa3-type cytochrome c oxidase from Rhodobacter sphaeroides.

Cytochrome c oxidase uses the free energy of oxygen reduction to establish a transmembrane proton gradient. The proton-conducting D-channel in this enzyme is the major input pathway for protons which go to the binuclear center for water formation ("chemical protons") and likely the only input pathway for protons that get translocated across the lipid membrane ("pumped protons"). The D-channel starts at an acidic residue near the protein surface (D132, Rhodobacter sphaeroides numbering) and leads to another acidic residue near the binuclear center. Recent studies have shown that mutants that introduce an additional acidic residue in the channel (N139D) have the remarkable effect of accelerating steady-state oxidase activity but completely eliminating proton pumping. In this work, an aspartic acid was introduced at the position of glycine 204, G204D, which is also within the D-channel, and the effects were examined. In contrast to N139D, the G204D mutation results in a dramatic decrease of the steady-state oxygen reductase activity (<2% of wild type) [Aagaard, A., and Brzezinski, P. (2001) FEBS Lett. 494, 157-160]. The residual activity is not coupled to the proton pump, and furthermore, in reconstituted vesicles the mutant enzyme exhibits a reverse respiration control ratio; i.e., the mutant oxidase activity is stimulated rather than inhibited when working against a protonmotive force. Hence, the mutant behaves very much like the D132N, which blocks proton uptake through the D-channel. Single-turnover experiments show that the rate-limiting step in the reaction of O2 with the fully reduced G204D mutant is the F --> O transition, similar to the D132N mutant. The block of the D-channel in the D132N mutant can be partly bypassed by biochemically removing subunit III from the enzyme, indicating that removal of the subunit reveals an alternate entrance for protons to the channel. However, this is not observed with the G204D mutant. This suggests that the cryptic entrance to the D-channel that is revealed by the removal of subunit III is between the levels of G204 and D132.

Arachidonic Acid↗
PubMed2006

Calculated proton uptake on anaerobic reduction of cytochrome C oxidase: is the reaction electroneutral?

Cytochrome c oxidase is a transmembrane proton pump that builds an electrochemical gradient using chemical energy from the reduction of O(2). Ionization states of all residues were calculated with Multi-Conformation Continuum Electrostatics (MCCE) in seven anaerobic oxidase redox states ranging from fully oxidized to fully reduced. One long-standing problem is how proton uptake is coupled to the reduction of the active site binuclear center (BNC). The BNC has two cofactors: heme a(3) and Cu(B). If the protein needs to maintain electroneutrality, then 2 protons will be bound when the BNC is reduced by 2 electrons in the reductive half of the reaction cycle. The effective pK(a)s of ionizable residues around the BNC are evaluated in Rhodobacter sphaeroides cytochrome c oxidase. At pH 7, only a hydroxide coordinated to Cu(B) shifts its pK(a) from below 7 to above 7 and so picks up a proton when heme a(3) and Cu(B) are reduced. Glu I-286, Tyr I-288, His I-334, and a second hydroxide on heme a(3) all have pK(a)s above 7 in all redox states, although they have only 1.6-3.5 DeltapK units energy cost for deprotonation. Thus, at equilibrium, they are protonated and cannot serve as proton acceptors. The propionic acids near the BNC are deprotonated with pK(a)s well below 7. They are well stabilized in their anionic state and do not bind a proton upon BNC reduction. This suggests that electroneutrality in the BNC is not maintained during the anaerobic reduction. Proton uptake on reduction of Cu(A), heme a, heme a(3), and Cu(B) shows approximately 2.5 protons bound per 4 electrons, in agreement with prior experiments. One proton is bound by a hydroxyl group in the BNC and the rest to groups far from the BNC. The electrochemical midpoint potential (E(m)) of heme a is calculated in the fully oxidized protein and with 1 or 2 electrons in the BNC. The E(m) of heme a shifts down when the BNC is reduced, which agrees with prior experiments. If the BNC reduction is electroneutral, then the heme a E(m) is independent of the BNC redox state.

Anaerobiosis↗
PubMed1997

Glutamate and aspartate as proton shuttles in mutants of carbonic anhydrase.

Maximal turnover rates for the hydration of CO2 and the depletion of 18O from CO2 catalyzed by carbonic anhydrase III (CA III) and carbonic anhydrase V (CA V) are limited by proton transfer involving zinc-bound water or hydroxide in the active site. We have investigated the capacity of glutamic and aspartic acids at position 64 in human CA III and murine CA V to act as proton shuttles in this pathway. The distance from the Calpha of position 64 to the zinc is near 9.5 A in the crystal structures of both CA III and CA V. Rates of intramolecular proton transfer between these proton shuttle groups and the zinc-bound water molecule were estimated as the predominant rate-contributing step in the catalytic turnover kcat in the hydration of CO2 measured by stopped flow and in the 18O exchange between CO2 and water measured by mass spectrometry. We found that both glutamate and aspartate residues at position 64 are efficient proton shuttles in HCA III. The rate constant for intramolecular proton transfer from either residue to zinc-bound hydroxide is 4 x 10(4) s-1, about 20-fold greater than that of the wild type which has lysine at position 64. When the active site residue Phe 198 in human CA III was replaced with Leu, measurement of catalysis showed that Glu 64 retained but Asp 64 lost its capacity to act as a proton shuttle. These observations were supported in studies of catalysis by murine CA V which contains Leu 198; here again, Glu 64 acted as a proton shuttle, but Asp 64 did not. Phe 198 in HCA III is thus a significant factor in the capacity of the active site to sustain proton transfer, possibly through its stabilization of hydrogen-bonded water bridges that enhance proton translocation from both Glu and Asp at position 64 to the zinc-bound hydroxide.

Animals↗
PubMed1998

Hydrogen bonding, solvent exchange, and coupled proton and electron transfer in the oxidation and reduction of redox-active tyrosine Y(Z) in Mn-depleted core complexes of photosystem II.

The redox-active tyrosines, Y(Z) and Y(D), of Photosystem II are oxidized by P680+ to the neutral tyrosyl radical. This oxidation thus involves the transfer of the phenolic proton as well as an electron. It has recently been proposed that tyrosine Y(Z) might replace the lost proton by abstraction of a hydrogen atom or a proton from a water molecule bound to the manganese cluster, thereby increasing the driving force for water oxidation. To compare and contrast with the intact system, we examine here, in a simplified Mn-depleted PSII core complex, isolated from a site-directed mutant of Synechocystis PCC 6803 lacking Y(D), the role of proton transfer in the oxidation and reduction of Y(Z). We show how the oxidation and reduction rates for Y(Z), the deuterium isotope effect on these rates, and the Y(Z)* - Y(Z) difference spectra all depend on pH (from 5.5 to 9.5). This simplified system allows examination of electron-transfer processes over a broader range of pH than is possible with the intact system and with more tractable rates. The kinetic isotope effect for the oxidation of P680+ by Y(Z) is maximal at pH 7.0 (3.64). It decreases to lower pH as charge recombination, which shows no deuterium isotope, starts to become competitive with Y(Z) oxidation. To higher pH, Y(Z) becomes increasingly deprotonated to form the tyrosinate, the oxidation of which at pH 9.5 becomes extremely rapid (1260 ms(-1)) and no longer limited by proton transfer. These observations point to a mechanism for the oxidation of Y(Z) in which the tyrosinate is the species from which the electron occurs even at lower pH. The kinetics of oxidation of Y(Z) show elements of rate limitation by both proton and electron transfer, with the former dominating at low pH and the latter at high pH. The proton-transfer limitation of Y(Z) oxidation at low pH is best explained by a gated mechanism in which Y(Z) and the acceptor of the phenolic proton need to form an electron/proton-transfer competent complex in competition with other hydrogen-bonding interactions that each have with neighboring residues. In contrast, the reduction of Y(Z)* appears not to be limited by proton transfer between pH 5.5 and 9.5. We also compare, in Mn-depleted Synechocystis PSII core complexes, Y(Z) and Y(D) with respect to solvent accessibility by detection of the deuterium isotope effect for Y(Z) oxidation and by 2H ESEEM measurement of hydrogen-bond exchange. Upon incubation of H2O-prepared PSII core complexes in D2O, the phenolic proton of Y(Z) is exchanged for a deuterium in less than 2 min as opposed to a t(1/2) of about 9 h for Y(D). In addition, we show that Y(D)* is coordinated by two hydrogen bonds. Y(Z)* shows more disordered hydrogen bonding, reflecting inhomogeneity at the site. With 2H ESEEM modulation comparable to that of Y(D)*, Y(Z)* would appear to be coordinated by two hydrogen bonds in a significant fraction of the centers.

Chlorophyll↗
PubMed2003

Protein side-chain motion and hydration in proton-transfer pathways. Results for cytochrome p450cam.

Proton-transfer reactions form an integral part of bioenergetics and enzymatic catalysis. The identification of proton-conducting pathways inside a protein is a key to understanding the mechanisms of biomolecular proton transfer. Proton pathways are modeled here as hydrogen bonded networks of proton-conducting groups, including proton-exchanging groups of amino acid side chains and bound water molecules. We focus on the identification of potential proton-conducting pathways inside a protein of known structure. However, consideration of the static structure alone is often not sufficient to detect suitable proton-transfer paths, leading, for example, from the protein surface to the active site buried inside the protein. We include dynamic fluctuations of amino acid side chains and water molecules into our analysis. To illustrate the method, proton transfer into the active site of cytochrome P450cam is studied. The cooperative rotation of amino acids and motion of water molecules are found to connect the protein surface to the molecular oxygen. Our observations emphasize the intrinsic dynamical nature of proton pathways where critical connections in the network may be transiently provided by mobile groups.

Camphor 5-Monooxygenase↗
PubMed2006

Calculation of vibronic couplings for phenoxyl/phenol and benzyl/toluene self-exchange reactions: implications for proton-coupled electron transfer mechanisms.

The vibronic couplings for the phenoxyl/phenol and the benzyl/toluene self-exchange reactions are calculated with a semiclassical approach, in which all electrons and the transferring hydrogen nucleus are treated quantum mechanically. In this formulation, the vibronic coupling is the Hamiltonian matrix element between the reactant and product mixed electronic-proton vibrational wavefunctions. The magnitude of the vibronic coupling and its dependence on the proton donor-acceptor distance can significantly impact the rates and kinetic isotope effects, as well as the temperature dependences, of proton-coupled electron transfer reactions. Both of these self-exchange reactions are vibronically nonadiabatic with respect to a solvent environment at room temperature, but the proton tunneling is electronically nonadiabatic for the phenoxyl/phenol reaction and electronically adiabatic for the benzyl/toluene reaction. For the phenoxyl/phenol system, the electrons are unable to rearrange fast enough to follow the proton motion on the electronically adiabatic ground state, and the excited electronic state is involved in the reaction. For the benzyl/toluene system, the electrons can respond virtually instantaneously to the proton motion, and the proton moves on the electronically adiabatic ground state. For both systems, the vibronic coupling decreases exponentially with the proton donor-acceptor distance for the range of distances studied. When the transferring hydrogen is replaced with deuterium, the magnitude of the vibronic coupling decreases and the exponential decay with distance becomes faster. Previous studies designated the phenoxyl/phenol reaction as proton-coupled electron transfer and the benzyl/toluene reaction as hydrogen atom transfer. In addition to providing insights into the fundamental physical differences between these two types of reactions, the present analysis provides a new diagnostic for differentiating between the conventionally defined hydrogen atom transfer and proton-coupled electron transfer reactions.

Electrons↗
PubMed2005

Proton translocation in monomolecular Langmuir-Blodgett films including 2-naphthol and 1,4-anthraquinone derivatives.

The scope of the present work is the investigation of proton transport through monomolecular Langmuir-Blodgett (LB) films. The films were formed from amphiphilic molecules: 2-naphtholo-6-sulfonamide of dodecylamine (N) and 1,4-anthraquinono-2 sulfonamide of dodecylamine (A). The 2-naphthol derivative can act as a proton donor due to excited state proton transfer (ESPT) and the 1,4-anthraquinone group can play the role of proton acceptor because of protonation of the reduced form if it is present. Absorption and emission spectra of LB films containing N and A were registered and separated into component bands. Individual absorption and emission peaks observed were assigned to given forms of chromophores. The behavior of different component bands reflects the state of anthraquinone dependent on proton concentration. A correlation of rate and efficiency of ESPT, with changes of the spectra of A, may be expected to yield information concerning the transport of protons from N to A. The influence of the donor-acceptor distance, sample humidity, film arrangement and the presence of protonophores (Gramicidin A) on proton transfer is studied. Our results indicate that the proton can be transported through the film but its concentration vanishes at the distance greater than 30 A. The efficiency of proton transfer depends strongly on water content, film structure and the presence of ion channels.

Anthraquinones↗