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PubMed2002

Usefulness of positron-emission tomographic images after proton therapy.

PURPOSE: To examine the positron emission tomography (PET) image obtained after proton irradiation and investigate the usefulness of the image for confirmation of the irradiated volume in proton radiotherapy (RT). METHODS AND MATERIALS: A homogenous phantom was irradiated separately by carbon-ion and proton beams and the images obtained were compared. The PET images of cancer patients just after proton RT were then taken after informed consent. RESULTS: In the PET image produced by carbon-ion beams, the high pixel counts in the image corresponded to the Bragg peak; however, in that produced by proton beams, they were visible throughout the entire track of the proton beams and were not related to the Bragg peak. The PET image of patients treated with proton RT was similar to that of the phantom experiment. CONCLUSION: The PET image after proton RT was different from that of carbon-ion RT. It was found that the PET image was very useful in proton RT to verify treatment planning.

Brain↗
PubMed1997

Calculation of relative biological effectiveness for proton beams using biological weighting functions.

PURPOSE: The microdosimetric weighting function approach is used widely for beam comparison studies. The suitability of this model to predict the relative biological effectiveness (RBE) of therapeutic proton beams was studied. The RBE(alpha) (i.e., linear approximation) dependence on the type of biological end point, initial proton energy, energy spread of the input proton beam, and depth of beam penetration was investigated. METHODS AND MATERIALS: Proton transport calculations for a proton energy range from 70 to 250 MeV were performed to obtain proton energy spectra at a given depth. The corresponding microdosimetric distributions of lineal energy were calculated. To these distributions the biological response function approach was applied to calculate RBE(alpha) the biological effectiveness based on a linear dose-response relationship. The early intestinal tolerance assessed by crypt regeneration in mice and the inactivation of V79 cells were taken as biological end points. RESULTS: The RBE(alpha) values approach about 1 in the plateau region and gradually increase with the proton penetration depth. In the center of the Bragg peak, at the maximum dose delivery, the values of RBE(alpha) range from 1.1 (250-MeV beam, early intestinal tolerance in mice) to 1.9 (70-MeV beam, Chinese hamster V79 cells in G1/S phase). Distal to the Bragg peak, where only a small fraction of dose is delivered, the RBE(alpha) was found to be even higher. For modulated proton beams we found an increasing RBE(alpha) with depth in the spread-out Bragg peak (SOBP). Values up to 1.37 at the distal end of the SOBP plateau (155-MeV beam, SOBP between 5.3 and 13.2 cm) were obtained. CONCLUSION: More experimental work on the determination of microdosimetric weighting functions is needed. The results of the presented calculations indicate that for therapy planning it may be necessary to account for a depth dependence on proton RBE, especially for lower energy.

Animals↗
PubMed2000

Mononucleotide gas-phase proton affinities as determined by the kinetic method.

The goal of this work is to determine the proton affinities of (deoxy)nucleoside 5'- and 3'-monophosphates (mononucleotides) using the kinetic method with fast atom bombardment mass spectrometry. The proton affinities of the (deoxy)nucleoside 5'- and 3'-monophosphates yielded the following trend: (deoxy)adenosine monophosphates > (deoxy)guanosine monophosphates > (deoxy)cytidine monophosphates >> deoxythymidine/uridine monophosphates. In all cases the proton affinity decreases or remains the same with the addition of the phosphate group from those values reported for nucleosides. The proton affinity is dependent on the location of the phosphate backbone (5'-vs. 3'-phosphates): the 3'-monophosphates have lower proton affinities than the 5'-monophosphates except for the thymidine/uridine monophosphates where the trend is reversed. Molecular modeling was utilized to determine if multiple protonation sites and intramolecular hydrogen bond formation would influence the proton affinity measurements. Semiempirical calculations of the proton affinities at various locations on each mononucleotide were performed and compared to the experimental results. The possible influence of intramolecular hydrogen bonding between the nucleobases and the phosphate group on the measured and calculated proton affinities is discussed.

Algorithms↗
PubMed2003

An NMR shielding model for protons above the plane of a carbonyl group.

Covalently bonded hydrogen nuclei located over the plane of a carbonyl group in a strong magnetic field experience magnetic shielding (or deshielding) that results from the combined effect of the magnetic anisotropy of the carbon-oxygen double bond and various other intramolecular shielding effects. GIAO-HF in Gaussian 98 was employed to calculate isotropic shielding values and to predict the net proton NMR shielding increment for a simple model system: the proximate proton of methane held in various positions over formaldehyde. The net shielding increments of the proximate proton of methane, plotted against its Cartesian coordinates relative to the center of the carbon-oxygen double bond, led to the development of a single empirical equation for predicting the NMR shielding experienced by a covalently bonded proton over the plane of a carbonyl group. The predictive capability of this equation has been validated by calculating the shielding increments of protons over the plane of a carbonyl group in known structures, using this as a correction to the chemical shift estimated by subtituent effects and comparing the result to experimentally observed chemical shifts. Shielding is predicted by this equation for protons located in the region from over the center of the carbon-oxygen double bond to beyond the carbon atom; deshielding is predicted for protons located above and beyond the oxygen atom. This prediction differs from those made by the long-held "shielding cone" model found in nearly every textbook on NMR, but is consistent with experimental observations. The algorithm for predicting the shielding increment for a proton over a carbonyl group can be used in a spreadsheet or incorporated into software that estimates chemical shifts using additive substituent constants or a database of structures. Its use can improve the accuracy of the estimated chemical shift of a proton in the vicinity of a carbon-oxygen double bond, and thus assist in spectral assignments and in correct structure determination.

Anisotropy↗
PubMed2000

Protonation state of methyltetrahydrofolate in a binary complex with cobalamin-dependent methionine synthase.

N5-Methyltetrahydrofolate (CH(3)-H(4)folate) donates a methyl group to the cob(I)alamin cofactor in the reaction catalyzed by cobalamin-dependent methionine synthase (MetH, EC 2.1.1.3). Nucleophilic displacement of a methyl group attached to a tertiary amine is a reaction without an obvious precedent in bioorganic chemistry. Activation of CH(3)-H(4)folate by protonation prior to transfer of the methyl group has been the favored mechanism. Protonation at N5 would lead to formation of an aminium cation, and quaternary amines such as 5,5-dimethyltetrahydropterin have been shown to transfer methyl groups to cob(I)alamin. Because CH(3)-H(4)folate is an enamine, protonation could occur either at N5 to form an aminium cation or on a conjugated carbon with formation of an iminium cation. We used (13)C distortionless enhancement by polarization transfer (DEPT) NMR spectroscopy to infer that CH(3)-H(4)folate in aqueous solution protonates at N5, not on carbon. CH(3)-H(4)folate must eventually protonate at N5 to form the product H(4)folate; however, this protonation could occur either upon formation of the binary enzyme-CH(3)-H(4)folate complex or later in the reaction mechanism. Protonation at N5 is accompanied by substantial changes in the visible absorbance spectrum of CH(3)-H(4)folate. We have measured the spectral changes associated with binding of CH(3)-H(4)folate to a catalytically competent fragment of MetH over the pH range from 5.5 to 8.5. These studies indicate that CH(3)-H(4)folate is bound in the unprotonated form throughout this pH range and that protonated CH(3)-H(4)folate does not bind to the enzyme. Our observations are rationalized by sequence homologies between the folate-binding region of MetH and dihydropteroate synthase, which suggest that the pterin ring is bound in the hydrophobic core of an alpha(8)beta(8) barrel in both enzymes. The results from these studies are difficult to reconcile with an S(N)2 mechanism for methyl transfer and suggest that the presence of the cobalamin cofactor is important for CH(3)-H(4)folate activation. We propose that protonation of N5 occurs after carbon-nitrogen bond cleavage, and we invoke a mechanism involving oxidative addition of Co(1+) to the N5-methyl bond to rationalize our results.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1992

Pathways of proton release in the bacteriorhodopsin photocycle.

The pH dependencies of the rate constants in the photocycles of recombinant D96N and D115N/D96N bacteriorhodopsins were determined from time-resolved difference spectra between 70 ns and 420 ms after photoexcitation. The results were consistent with the model suggested earlier for proteins containing D96N substitution: BR hv----K----L----M1----M2----BR. Only the M2----M1 back-reaction was pH-dependent: its rate increased with increasing [H+] between pH 5 and 8. We conclude from quantitative analysis of this pH dependency that its reverse, the M1----M2 reaction, is linked to the release of a proton from a group with a pKa = 5.8. This suggests a model for wild-type bacteriorhodopsin in which at pH greater than 5.8 the transported proton is released on the extracellular side from this as yet unknown group and on the 100-microseconds time scale, but at pH less than 5.8, the proton release occurs from another residue and later in the photocycle most likely directly from D85 during the O----BR reaction. We postulate, on the other hand, that proton uptake on the cytoplasmic side will be by D96 and during the N----O reaction regardless of pH. The proton kinetics as measured with indicator dyes confirmed the unique prediction of this model: at pH greater than 6, proton release preceded proton uptake, but at pH less than 6, the release was delayed until after the uptake. The results indicated further that the overall M1----M2 reaction includes a second kinetic step in addition to proton release; this is probably the earlier postulated extracellular-to-cytoplasmic reorientation switch in the proton pump.

Bacteriorhodopsins↗
PubMed1982

Proton release during the pre-steady-state oxidation of aldehydes by aldehyde dehydrogenase. Evidence for a rate-limiting conformational change.

A transient release of protons with an amplitude corresponding to one proton per active site has been observed for the oxidation of propionaldehyde, acetaldehyde, and benzaldehyde by sheep liver cytoplasmic aldehyde dehydrogenase at pH 7.6 with phenol red as indicator. At saturating substrate levels, the rate constants for the proton burst are in each case the same, and for acetaldehyde and propionaldehyde show the same dependence on the concentrations of the substrates, as the rate constants for the transient production of NADH reported previously [MacGibbon, A.K.H., Blackwell, L.F., & Buckley, P.D. (1977) Biochem. J. 167, 469-477]. Although, with propionaldehyde as a substrate, a full proton burst is also observed at pH 6.0, no proton burst is observed at pH 9.0. For 4-nitrobenzaldehyde, there is no burst in NADH production, but a burst in proton release is observed, showing that proton release precedes hydride transfer. No protons were released during the binding of the substrate analogues acetone and chloral hydrate nor on reaction of the enzyme with the inhibitor tetraethylthiuram disulfide (disulfiram). A model is proposed in which the rate-limiting step in the pre-steady-state phase of the reaction is a conformational change which occurs after the binding of aldehydes to the enzyme. As a result of the conformational change, the environment of a functional group on the enzyme, which initially has a pKa of about 8.5, is perturbed to give a final pKa value for the group of less than 5. Computer simulations were used to show that the model accurately reproduces all of the experimental data. The lack of observation of a second transient proton release, as required by the overall stoichiometry, argues that its release occurs in a slow step prior to NADH dissociation.

Acetaldehyde↗
PubMed1988

Hydration of CO2 by carbonic anhydrase: intramolecular proton transfer between Zn2+-bound H2O and histidine 64 in human carbonic anhydrase II.

The energy barrier for the intramolecular proton transfer between zinc-bound water and His 64 in the active site of human carbonic anhydrase II (HCA II) has been studied at the partial retention of diatomic differential overlap (PRDDO) level. The most important stabilizing factor for the intramolecular proton transfer is the zinc ion, which lowers the pKa of zinc-bound water and electrostatically repels the proton. The energy barrier of 127.5 kcal/mol for proton transfer between a water dimer is completely removed in the presence of the zinc ion. The zinc ligands, which donate electrons to the zinc ion, raise the barrier slightly to 34 kcal/mol for a 4-coordinated zinc complex including three imidazole ligands from His 94, His 96, and His 119 and to 54 kcal/mol for the 5-coordinated zinc complex including the fifth water ligand. A few model calculations indicate that these energy barriers are expected to be reduced to within experimental range (approximately 10 kcal/mol) when large basis set, correlation energies, and molecular dynamics are considered. The proton-transfer group, which functions as proton receiver in the intramolecular proton transfer, helps to attract the proton; and the partially ordered active site water molecules are important for proton relay function.

Binding Sites↗
PubMed2003

Proton transfer reactions associated with the reaction of the fully reduced, purified cytochrome C oxidase with molecular oxygen and ferricyanide.

A study is presented on proton transfer associated with the reaction of the fully reduced, purified bovine heart cytochrome c oxidase with molecular oxygen or ferricyanide. The proton consumption associated with aerobic oxidation of the four metal centers changed significantly with pH going from approximately 3.0 H(+)/COX at pH 6.2-6.3 to approximately 1.2 H(+)/COX at pH 8.0-8.5. Rereduction of the metal centers was associated with further proton uptake which increased with pH from approximately 1.0 H(+)/COX at pH 6.2-6.3 to approximately 2.8 H(+)/COX at pH 8.0-8.5. Anaerobic oxidation of the four metal centers by ferricyanide resulted in the net release of 1.3-1.6 H(+)/COX in the pH range 6.2-8.2, which were taken up by the enzyme on rereduction of the metal centers. The proton transfer elicited by ferricyanide represents the net result of deprotonation/protonation reactions linked to anaerobic oxidoreduction of the metal centers. Correction for the ferricyanide-induced pH changes of the proton uptake observed in the oxidation and rereduction phase of the reaction of the reduced oxidase with oxygen gave a measure of the proton consumption in the reduction of O(2) to 2H(2)O. The results show that the expected stoichiometric proton consumption of 4H(+) in the reduction of O(2) to 2H(2)O is differently associated, depending on the actual pH, with the oxidation and reduction phase of COX. Two H(+)/COX are initially taken up in the reduction of O(2) to two OH(-) groups bound to the binuclear Fe a(3)-Cu(B) center. At acidic pHs the third and fourth protons are also taken up in the oxidative phase with formation of 2H(2)O. At alkaline pHs the third and fourth protons are taken up with formation of 2H(2)O only upon rereduction of COX.

Animals↗
PubMed2002

Kinetics of proton uptake and dye binding by photoactive yellow protein in wild type and in the E46Q and E46A mutants.

We studied the kinetics of proton uptake and release by photoactive yellow protein (PYP) from Ectothiorhodospira halophila in wild type and the E46Q and E46A mutants by transient absorption spectroscopy with the pH-indicator dyes bromocresol purple or cresol red in unbuffered solution. In parallel, we investigated the kinetics of chromophore protonation as monitored by the rise and decay of the blue-shifted state I(2) (lambda(max) = 355 nm). For wild type the proton uptake kinetics is synchronized with the fast phase of I(2) formation (tau = 500 micros at pH 6.2). The transient absorption signal from the dye also contains a slower component which is not due to dye deprotonation but is caused by dye binding to a hydrophobic patch that is transiently exposed in the structurally changed and partially unfolded I(2) intermediate. This conclusion is based on the wavelength, pH, and concentration dependence of the dye signal and on dye measurements in the presence of buffer. SVD analysis, moreover, indicates the presence of two components in the dye signal: protonation and dye binding. The dye binding has a rise time of about 4 ms and is coupled kinetically with a transition between two I(2) intermediates. In the mutant E46Q, which lacks the putative internal proton donor E46, the formation of I(2) is accelerated, but the proton uptake kinetics remains kinetically coupled to the fast phase of I(2) formation (tau = 100 micros at pH 6.3). For this mutant the protein conformational change, as monitored by the dye binding, occurs with about the same time constant as in wild type but with reduced amplitude. In the alkaline form of the mutant E46A the formation of the I(2)-like intermediate is even faster as is the proton uptake (tau = 20 micros at pH 8.3). No dye binding occurred in E46A, suggesting the absence of a conformational change. In all of the systems proton release is synchronized with the decay of I(2). Our results support mechanisms in which the chromophore of PYP is protonated directly from the external medium rather than by the internal donor E46.

Bacterial Proteins↗
PubMed2003

Proton transfer reactions in the F86D and F86E mutants of pharaonis phoborhodopsin (sensory rhodopsin II).

pharaonis phoborhodopsin (ppR, also called pharaonis sensory rhodopsin II, psRII), a negative phototaxis receptor of Natronobacterium pharaonis, can use light to pump a proton in the absence of its transducer protein. However, the pump activity is much lower than that of the light-driven proton-pump bacteriorhodopsin (BR). ppR's pump activity is known to be increased in a mutant protein, in which Phe86 is replaced with Asp (F86D). Phe86 is the amino acid residue corresponding to Asp96 in BR, and we expect that Asp86 plays an important role in the proton transfer at the highly hydrophobic cytoplasmic domain of the F86D mutant ppR. In this article, we studied protein structural changes and proton transfer reactions during the photocycles of the F86D and F86E mutants in ppR by means of Fourier transform infrared (FTIR) spectroscopy and photoelectrochemical measurements using a tin oxide (SnO2) electrode. FTIR spectra of the unphotolyzed state and the K and M intermediates are very similar among F86D, F86E, and the wild type. Asp86 or Glu86 is protonated in F86D or F86E, respectively, and the pK(a) > 9. During the photocycle, the pK(a) is lowered and deprotonation of Asp86 or Glu86 is observed. Detection of both deprotonation of Asp86 or Glu86 and concomitant reprotonation of the 13-cis chromophore implies the presence of a proton channel between position 86 and the Schiff base. However, the photoelectrochemical measurements revealed proton release presumably from Asp86 or Glu86 to the cytoplasmic aqueous phase in the M state. This indicates that the ppR mutants do not have the BR-like mechanism that conducts a proton uniquely from Asp86 or Glu86 (Asp96 in BR) to the Schiff base, which is possible in BR by stepwise protein structural changes at the cytoplasmic side. In ppR, there is a single open structure at the cytoplasmic side (the M-like structure), which is shown by the lack of the N-like protein structure even in F86D and F86E at alkaline pH. Therefore, it is likely that a proton can be conducted in either direction, the Schiff base or the bulk, in the open M-like structure of F86D and F86E.

Amino Acid Substitution↗
PubMed2005

Kinetics of proton-linked flavin conformational changes in p-hydroxybenzoate hydroxylase.

p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction. Two structural features are coupled to control the reactivity of PHBH with NADPH: a proton-transfer network that allows protons to be passed between the sequestered active site and solvent and a flavin that adopts two positions: "in", where the flavin is near pOHB, and "out", where the flavin is near NADPH. PHBH uses the proton-transfer network to test for the presence of a suitable aromatic substrate before allowing the flavin to adopt the NADPH-accessible conformation. In this work, kinetic analysis of the His72Asn mutant, with a disrupted proton-transfer network, showed that flavin movement could occur in the presence or absence of NADPH but that NADPH stimulated movement to the reactive conformation required for hydride transfer. Substrate and solvent isotope effects on the transient kinetics of reduction of the His72Asn mutant showed that proton transfer was linked to flavin movement and that the conformational change occurred in a step separate from that of hydride transfer. Proton transfers during the reductive half-reaction were observed directly in the wild-type enzyme by performing experiments in the presence of a fluorescent pH-indicator dye in unbuffered solutions. NADPH binding caused rapid proton release from the enzyme, followed by proton uptake after flavin reduction. Solvent and substrate kinetic isotope effects showed that proton-coupled flavin movement and reduction also occurred in different steps in wild-type PHBH. These results allow a detailed kinetic scheme to be proposed for the reductive half-reaction of the wild-type enzyme. Three kinetic models considered for substrate-induced isomerization are analyzed in the Appendix.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed2005

An elementary reaction step of the proton pump is revealed by mutation of tryptophan-164 to phenylalanine in cytochrome c oxidase from Paracoccus denitrificans.

Cytochrome c oxidase couples reduction of dioxygen to water to translocation of protons over the inner mitochondrial or bacterial membrane. A likely proton acceptor for pumped protons is the Delta-propionate of heme a(3), which may receive the proton via water molecules from a conserved glutamic acid (E278 in subunit I of the Paracoccus denitrificans enzyme) and which receives a hydrogen bond from a conserved tryptophan, W164. Here, W164 was mutated to phenylalanine (W164F) to further explore the role of the heme a(3) Delta-propionate in proton translocation. FTIR spectroscopy showed changes in vibrations possibly attributable to heme propionates, and the midpoint redox potential of heme a(3) decreased by approximately 50 mV. The reaction of the oxidized W164F enzyme with hydrogen peroxide yielded substantial amounts of the intermediate F' even at high pH, which suggests that the mutation rearranges the local electric field in the binuclear center that controls the peroxide reaction. The steady-state proton translocation stoichiometry of the W164F enzyme dropped to approximately 0.5 H(+)/e(-) in cells and reconstituted proteoliposomes. Time-resolved electrometric measurements showed that when the fully reduced W164F enzyme reacted with O(2), the membrane potential generated in the fast phase of this reaction was far too small to account either for full proton pumping or uptake of a substrate proton from the inside of the proteoliposomes. Time-resolved optical spectroscopy showed that this fast electrometric phase occurred with kinetics corresponding to the transition from state A to P(R), whereas the subsequent transition to the F state was strongly delayed. This is due to a delay of reprotonation of E278 via the D-pathway, which was confirmed by observation of a slowed rate of Cu(A) oxidation and which explains the small amplitude of the fast charge transfer phase. Surprisingly, the W164F mutation thus mimics a weak block of the D-pathway, which is interpreted as an effect on the side chain isomerization of E278. The fast charge translocation may be due to transfer of a proton from E278 to a "pump site" above the heme groups and is likely to occur also in wild-type enzyme, though not distinguished earlier due to the high-amplitude membrane potential formation during the P(R)--> F transition.

Amino Acid Substitution↗
PubMed1996

Base-catalysis of imino proton exchange in DNA: effects of catalyst upon DNA structure and dynamics.

Characterization of the kinetics and energetics of base-pair opening in nucleic acids relies upon measurements of the rates of exchange of imino protons with water protons at high concentrations of the exchange catalyst. Under these conditions, the exchange catalyst may affect structural or dynamic properties of the nucleic acid molecule and thus, limit the significance of the exchange data. To address this problem, we have used NMR spectroscopy to characterize the effects of a catalyst of imino proton exchange, namely, ammonia upon the structure and dynamics of the self-complementary DNA dodecamer [d(CGCAGATCTGCG)]2. The changes in structure were monitored in proton NOESY and DQF-COSY experiments and in phosphorus spectra at 15 degrees C and at ammonia concentrations ranging from 0.002 to 0.5 M. The results indicate that ammonia induces subtle changes in the solution conformation of the dodecamer, but the overall structure is maintained close to the B-type DNA structure. However, the relaxation rates (i.e., transverse, longitudinal, and cross-relaxation rates) of several non-exchangeable protons were found to increase by approximately 50% upon changing ammonia concentration from 0.002 to 0.5 M. The increases were comparable for all protons investigated suggesting that they originate from an ammonia-induced increase in the overall correlation time of the DNA dodecamer. Numerical analysis revealed that the catalyst-induced enhancements in proton relaxation can alter significantly the calculated values of the exchange rates of imino protons, especially those obtained from measurements of the line widths of these proton resonances.

Alkalies↗
PubMed1998

Role of the pathway through K(I-362) in proton transfer in cytochrome c oxidase from R. sphaeroides.

In this study we have combined the use of site-directed mutants with time-resolved optical absorption spectroscopy to investigate the role of the protonatable subunit-I residues lysine-362 (K(I-362)) and threonine-359 (T(I-359)) in cytochrome c oxidase from Rhodobacter sphaeroides in electron and proton transfer. These residues have been proposed to be part of a proton-transfer pathway in cytochrome oxidases from Paracoccus denitrificans and bovine heart. Mutation of K(I-362) and T(I-359) to methionine and alanine, respectively, results in reduction of the overall turnover activities to <2% and approximately 35%, respectively, of those in the wild-type enzyme. The results show that in the absence of dioxygen, electron transfer between hemes a3 and a with a time constant of approximately 3 micros, not coupled to protonation reactions, is not affected in the mutant enzymes. However, the slower electron transfer between hemes a3 and a, coupled to proton release with a time constant of approximately 3 ms (at pH 9.0) is impaired in the KM(I-362) and TA(I-359) mutant enzymes. This is consistent with the slow reduction rate of heme a3 in the oxidized KM(I-362) enzyme because in the wild-type enzyme reduction of heme a3 is coupled to proton uptake. On the other hand, when reacting with O2, both the wild-type and mutant fully reduced enzymes become oxidized in approximately 5 ms, and proton uptake on this time scale is not affected. Hence, the results indicate that the KM(I-362) mutant enzyme is inactive because the proton-transfer pathway through K(I-362) and T(I-359) is involved in proton uptake during reduction of the oxidized binuclear center. Proton uptake during oxidation of the fully reduced enzyme takes place through a different pathway [through E(I-286) (Adelroth, P., et al. (1997) Biochemistry 36, 13824-13829)].

Animals↗
PubMed2005

Proton binding to humic acids from organic amendments and amended soils by the NICA-Donnan model.

The acid-base properties of humic acids (HAs) are known to significantly affect the acid-base buffering capacity of soils, thus having a marked influence on the speciation of cations in the soil solid and liquid phases. Detailed information on the proton binding behavior of humic-like acids (HALs) from organic amendments and humic acids (HAs) from amended soils is, therefore, of intrinsic interest for the evaluation of the agronomic efficacy and environmental impact of soil amendment. In this work, the acid-base properties of HLAs isolated from sewage sludge (SS) and municipal solid waste compost (MSWC), and HAs isolated from soils amended with either SS or MSWC and the corresponding nonamended control soils were investigated by potentiometric titrations at various ionic strengths (0.01, 0.05, 0.1, and 0.3 M) over the pH range from 3.5 to 10.5. The nonideal competitive adsorption (NICA)-Donnan model that describes proton binding by two classes of binding sites with low and high proton affinity, i.e., carboxylic- and phenolic-type groups, was fit to titration data, and a set of fitting parameters was obtained for each HLA and HA sample. The NICA-Donnan model successfully described the shapes of the titration curves, and highlighted substantial differences in site density and proton-binding affinity between the HLAs and HAs examined. With respect to the nonamended control soil HAs, SS-HLA and MSWC-HLA were characterized by smaller carboxylic-type and phenolic-type group contents, larger affinities for proton binding by the carboxylic-type groups, and smaller affinities for proton binding by the phenolic-type groups. Amendment with SS and MSWC determined a number of modifications in soil HAs, including decrease of acidic functional group contents, slight increase of proton affinity of carboxylic-type groups, and slight decrease of the affinities for proton binding by phenolic-type groups. These effects were more evident in the HA fraction from the SS-amended soil than in the HA fraction from the MSWC-amended soil. Thus, both organic amendments examined can be a considered as a valuable source of organic matter for soil. However, MSWC appears to be an amendment of greater quality producing a smaller impact than SS on proton-binding behavior of soil HA.

Binding Sites↗
PubMed2001

Protonation of platinated adenine nucleobases. Gas phase vs condensed phase picture.

Protonation of adenine carrying a Pt(II) moiety either at N7, N3, or N1 is possible in solution, but the site of protonation is influenced by the location of the Pt(II) electrophile and to some extent also by the overall charge of the metal entity (+2, +1, 0, -1), hence the other ligands (NH(3), Cl(-), OH(-)) bound to Pt(II). Quantum chemical calculations based on density functional theory (DFT) have been carried out for intrinsic protonation energies of adenine complexes carrying the following Pt(II) species at either of the three ring N atoms: [Pt(NH(3))(3)](2+) (1), trans- [Pt(NH(3))(2)Cl](+) (2a), cis-[Pt(NH(3))(2)Cl](+) (2b), trans-[Pt(NH(3))(2)Cl(2)] (3a), cis-[Pt(NH(3))Cl(2)] (3b), [PtCl(3)](-) (4), trans-[Pt(NH(3))(2)OH](+) (5a), cis-[Pt(NH(3))(2)(OH)](+) (5b), trans-[Pt(NH(3))(OH)(2)] (6a), cis-[Pt(NH(3))(OH)(2)] (6b), and [Pt(OH)(3)](-) (7). The data have been compared with results derived from solution studies (water) and X-ray crystallography, whenever available. The electrostatic effects associated with the charge of the metal entity have the major influence on the calculated intrinsic (gas phase) proton affinities, unlike the condensed phase data. Nevertheless, the relative gas phase trends correlate surprisingly well with condensed phase data; i.e., variation of the pK(a) values measured in solution is consistent with the calculated gas phase protonation energies. In addition to a systematic study of the ring proton affinities, proton transfer processes within the platinated adenine species were often observed when investigating Pt adducts with OH(-) ligands, and they are discussed in more detail. To the best of our knowledge, this is the first study attempting to find a systematic correlation between gas phase and condensed phase data on protonation of metalated nucleobases. The gas phase data provide a very useful complement to the condensed phase and X-ray experiments, showing that the gas phase studies are capable of valuable predictions and contribute to our understanding of the solvent and counterion effects on metal-assisted proton shift processes.

Adenine Nucleotides↗
PubMed2004

Key role of electrostatic interactions in bacteriorhodopsin proton transfer.

The first proton transport step following photon absorption in bacteriorhodopsin is from the 13-cis retinal Schiff base to Asp85. Configurational and energetic determinants of this step are investigated here by performing quantum mechanical/molecular mechanical minimum-energy reaction-path calculations. The results suggest that retinal can pump protons when in the 13-cis, 15-anti conformation but not when 13-cis, 15-syn. Decomposition of the proton transfer energy profiles for various possible pathways reveals a conflict between the effect of the intrinsic proton affinities of the Schiff base and Asp85, which favors the neutral, product state (i.e., with Asp85 protonated), with the mainly electrostatic interaction between the protein environment with the reacting partners, which favors the ion pair reactant state (i.e., with retinal protonated). The rate-limiting proton-transfer barrier depends both on the relative orientations of the proton donor and acceptor groups and on the pathway followed by the proton; depending on these factors, the barrier may arise from breaking and forming of hydrogen bonds involving the Schiff base, Asp85, Asp212, and water w402, and from nonbonded interactions involving protein groups that respond to the charge rearrangements in the Schiff base region.

Bacteriorhodopsins↗