Potentiation of proton transfer function by electrostatic interactions in photosynthetic reaction centers from Rhodobacter sphaeroides: First results from site-directed mutation of the H subunit.

The x-ray crystallographic structure of the photosynthetic reaction center (RC) has proven critical in understanding biological electron transfer processes. By contrast, understanding of intraprotein proton transfer is easily lost in the immense richness of the details. In the RC of Rhodobacter (Rb.) sphaeroides, the secondary quinone (QB) is surrounded by amino acid residues of the L subunit and some buried water molecules, with M- and H-subunit residues also close by. The effects of site-directed mutagenesis upon RC turnover and quinone function have implicated several L-subunit residues in proton delivery to QB, although some species differences exist. In wild-type Rb. sphaeroides, Glu L212 and Asp L213 represent an inner shell of residues of particular importance in proton transfer to QB. Asp L213 is crucial for delivery of the first proton, coupled to transfer of the second electron, while Glu L212, possibly together with Asp L213, is necessary for delivery of the second proton, after the second electron transfer. We report here the first study, by site-directed mutagenesis, of the role of the H subunit in QB function. Glu H173, one of a cluster of strongly interacting residues near QB, including Asp L213, was altered to Gln. In isolated mutant RCs, the kinetics of the first electron transfer, leading to formation of the semiquinone, QB-, and the proton-linked second electron transfer, leading to the formation of fully reduced quinol, were both greatly retarded, as observed previously in the Asp L213 --> Asn mutant. However, the first electron transfer equilibrium, QA-QB <==> QAQB-, was decreased, which is opposite to the effect of the Asp L213 --> Asn mutation. These major disruptions of events coupled to proton delivery to QB were largely reversed by the addition of azide (N3-). The results support a major role for electrostatic interactions between charged groups in determining the protonation state of certain entities, thereby controlling the rate of the second electron transfer. It is suggested that the essential electrostatic effect may be to "potentiate" proton transfer activity by raising the pK of functional entities that actually transfer protons in a coupled fashion with the second electron transfer. Candidates include buried water (H3O+) and Ser L223 (serine-OH2+), which is very close to the O5 carbonyl of the quinone.

Expression cloning and functional characterization of the kidney cortex high-affinity proton-coupled peptide transporter.

The presence of a proton-coupled electrogenic high-affinity peptide transporter in the apical membrane of tubular cells has been demonstrated by microperfusion studies and by use of brush border membrane vesicles. The transporter mediates tubular uptake of filtered di- and tripeptides and aminocephalosporin antibiotics. We have used expression cloning in Xenopus laevis oocytes for identification and characterization of the renal high-affinity peptide transporter. Injection of poly(A)+ RNA isolated from rabbit kidney cortex into oocytes resulted in expression of a pH-dependent transport activity for the aminocephalosporin antibiotic cefadroxil. After size fractionation of poly(A)+ RNA the transport activity was identified in the 3.0- to 5.0-kb fractions, which were used for construction of a cDNA library. The library was screened for expression of cefadroxil transport after injection of complementary RNA synthesized in vitro from different pools of clones. A single clone (rPepT2) was isolated that stimulated cefadroxil uptake into oocytes approximately 70-fold at a pH of 6.0. Kinetic analysis of cefadroxil uptake expressed by the transporter's complementary RNA showed a single saturable high-affinity transport system shared by dipeptides, tripeptides, and selected amino-beta-lactam antibiotics. Electrophysiological studies established that the transport activity is electrogenic and affected by membrane potential. Sequencing of the cDNA predicts a protein of 729 amino acids with 12 membrane-spanning domains. Although there is a significant amino acid sequence identity (47%) to the recently cloned peptide transporters from rabbit and human small intestine, the renal transporter shows distinct structural and functional differences.

Efflux pump of the proton antiporter family confers low-level fluoroquinolone resistance in Mycobacterium smegmatis.

Due to the resurgence of tuberculosis and the emergence of multidrug-resistant strains, fluoroquinolones (FQ) are being used in selected tuberculosis patients, but FQ-resistant strains of Mycobacterium tuberculosis have rapidly begun to appear. The mechanisms involved in FQ resistance need to be elucidated if the effectiveness of this class of antibiotics is to be improved and prolonged. By using the rapid-growing Mycobacterium smegmatis as a model genetic system, a gene was selected that confers low-level FQ resistance when present on a multicopy plasmid. This gene, lfrA, encodes a putative membrane efflux pump of the major facilitator family, which appears to recognize the hydrophilic FQ, ethidium bromide, acridine, and some quaternary ammonium compounds. It is homologous to qacA from Staphylococcus aureus, tcmA, of Streptomyces glaucescens, and actII and mmr, both from Streptomyces coelicoler. Increased expression of lfrA augments the appearance of subsequent mutations to higher-level FQ resistance.

Proton transport by a bacteriorhodopsin mutant, aspartic acid-85-->asparagine, initiated in the unprotonated Schiff base state.

At alkaline pH the bacteriorhodopsin mutant D85N, with aspartic acid-85 replaced by asparagine, is in a yellow form (lambda max approximately 405 nm) with a deprotonated Schiff base. This state resembles the M intermediate of the wild-type photocycle. We used time-resolved methods to show that this yellow form of D85N, which has an initially unprotonated Schiff base and which lacks the proton acceptor Asp-85, transports protons in the same direction as wild type when excited by 400-nm flashes. Photoexcitation leads in several milliseconds to the formation of blue (630 nm) and purple (580 nm) intermediates with a protonated Schiff base, which decay in tens of seconds to the initial state (400 nm). Experiments with pH indicator dyes show that at pH 7, 8, and 9, proton uptake occurs in about 5-10 ms and precedes the slow release (seconds). Photovoltage measurements reveal that the direction of proton movement is from the cytoplasmic to the extracellular side with major components on the millisecond and second time scales. The slowest electrical component could be observed in the presence of azide, which accelerates the return of the blue intermediate to the initial yellow state. Transport thus occurs in two steps. In the first step (milliseconds), the Schiff base is protonated by proton uptake from the cytoplasmic side, thereby forming the blue state. From the pH dependence of the amplitudes of the electrical and photocycle signals, we conclude that this reaction proceeds in a similar way as in wild type--i.e., via the internal proton donor Asp-96. In the second step (seconds) the Schiff base deprotonates, releasing the proton to the extracellular side.

Features of MotA proton channel structure revealed by tryptophan-scanning mutagenesis.

The MotA protein of Escherichia coli is a component of the flagellar motors that functions in transmembrane proton conduction. Here, we report several features of MotA structure revealed by use of a mutagenesis-based approach. Single tryptophan residues were introduced at many positions within the four hydrophobic segments of MotA, and the effects on function were measured. Function was disrupted according to a periodic pattern that implies that the membrane-spanning segments are alpha-helices and that identifies the lipid-facing parts of each helix. The results support a hypothesis for MotA structure and mechanism in which water molecules form most of the proton-conducting pathway. The success of this approach in studying MotA suggests that it could be useful in structure-function studies of other integral membrane proteins.

Experimental evidence for hydrogen-bonded network proton transfer in bacteriorhodopsin shown by Fourier-transform infrared spectroscopy using azide as catalyst.

Experimental evidence for proton transfer via a hydrogen-bonded network in a membrane protein is presented. Bacteriorhodopsin's proton transfer mechanism on the proton uptake pathway between Asp-96 and the Schiff base in the M-to-N transition was determined. The slowdown of this transfer by removal of the proton donor in the Asp-96-->Asn mutant can be accelerated again by addition of small weak acid anions such as azide. Fourier-transform infrared experiments show in the Asp-96-->Asn mutant a transient protonation of azide bound to the protein in the M-to-N transition and, due to the addition of azide, restoration of the IR continuum band changes as seen in wild-type bR during proton pumping. The continuum band changes indicate fast proton transfer on the uptake pathway in a hydrogen-bonded network for wild-type bR and the Asp-96-->Asn mutant with azide. Since azide is able to catalyze proton transfer steps also in several kinetically defective bR mutants and in other membrane proteins, our finding might point to a general element of proton transfer mechanisms in proteins.

The photoreceptor sensory rhodopsin I as a two-photon-driven proton pump.

Proton translocation experiments with intact cells of Halobacterium salinarium overproducing sensory rhodopsin I (SRI) revealed transport activity of SRI in a two-photon process. The vectoriality of proton translocation depends on pH, being outwardly directed above, and inwardly directed below, pH 5.7. Activation of the transport cycle requires excitation of the initial dark state of SRI, SRI590, to form the intermediate SRI380. Action spectra identify the photocycle intermediates SRI380 and SRI520 as the two photochemically reactive species in the outwardly directed transport process. As shown by flash photolysis experiments, SRI520 undergoes a so-far unknown photochemical reaction to SRI380 with a half-time of <200 micros. Mutation of SRI residue Asp-76, the residue which is equivalent to the proton acceptor Asp-85 in bacteriorhodopsin, to asparagine leads to inactivation of proton translocation. This demonstrates that the underlying mechanisms of proton transport in both retinal proteins share similar features. However, SRI is to our knowledge the first case where photochemical reactions between two thermally unstable photoproducts of a retinal protein constitute a catalytic ion transport cycle.

Simulation of the secondary electrons energy deposition produced by proton beams in PMMA: influence of the target electronic excitation description

We have studied the radial dependence of the energy deposition of the secondary electron generated by swift proton beams incident with energies T = 50 keV–5 MeV on poly(methylmethacrylate) (PMMA). Two different approaches have been used to model the electronic excitation spectrum of PMMA through its energy loss function (ELF), namely the extended-Drude ELF and the Mermin ELF. The singly differential cross section and the total cross section for ionization, as well as the average energy of the generated secondary electrons, show sizeable differences at T ⩽ 0.1 MeV when evaluated with these two ELF models. In order to know the radial distribution around the proton track of the energy deposited by the cascade of secondary electrons, a simulation has been performed that follows the motion of the electrons through the target taking into account both the inelastic interactions (via electronic ionizations and excitations as well as electron-phonon and electron trapping by polaron creation) and the elastic interactions. The radial distribution of the energy deposited by the secondary electrons around the proton track shows notable differences between the simulations performed with the extended-Drude ELF or the Mermin ELF, being the former more spread out (and, therefore, less peaked) than the latter. The highest intensity and sharpness of the deposited energy distributions takes place for proton beams incident with T ~ 0.1–1 MeV. We have also studied the influence in the radial distribution of deposited energy of using a full energy distribution of secondary electrons generated by proton impact or using a single value (namely, the average value of the distribution); our results show that differences between both simulations become important for proton energies larger than ~0.1 MeV. The results presented in this work have potential applications in materials science, as well as hadron therapy (due to the use of PMMA as a tissue phantom) in order to properly consider the generation of electrons by proton beams and their subsequent transport and energy deposition through the target in nanometric scales.

Mécanismes de transfert de proton d’une réaction acido-basique en phase aqueuse : une étude ab-initio

Les réactions de transfert de proton se retrouvent abondamment dans la nature et sont des processus cruciaux dans plusieurs réactions chimiques et biologiques, qui se produisent souvent en milieu aqueux. Les mécanismes régissant ces échanges de protons sont complexes et encore mal compris, suscitant un intérêt des chercheurs en vue d’une meilleure compréhension fondamentale du processus de transfert. Le présent manuscrit présente une étude mécanistique portant sur une réaction de transfert de proton entre un acide (phénol fonctionnalisé) et une base (ion carboxylate) en phase aqueuse. Les résultats obtenus sont basés sur un grand nombre de simulations de dynamique moléculaire ab-initio réalisées pour des systèmes de type « donneur-pont-accepteur », où le pont se trouve à être une unique molécule d’eau, permettant ainsi l’élaboration d’un modèle cinétique détaillé pour le système étudié. La voie de transfert principalement observée est un processus ultra-rapide (moins d’une picoseconde) passant par la formation d’une structure de type « Eigen » (H9O4+) pour la molécule d’eau pontante, menant directement à la formation des produits. Une seconde structure de la molécule d’eau pontante est également observée, soit une configuration de type « Zündel » (H5O2+) impliquant l’accepteur de proton (l’ion carboxylate) qui semble agir comme un cul-de-sac pour la réaction de transfert de proton.

Measurements of π±, K±, K0s, Λ and proton production in proton–carbon interactions at 31 GeV/c with the NA61/SHINE spectrometer at the CERN SPS

Measurements of hadron production in p+C interactions at 31 GeV/c are performed using the NA61/SHINE spectrometer at the CERN SPS. The analysis is based on the full set of data collected in 2009 using a graphite target with a thickness of 4% of a nuclear interaction length. Inelastic and production cross sections as well as spectra of π±, K±, p, K0s and Λ are measured with high precision. These measurements are essential for improved calculations of the initial neutrino fluxes in the T2K long-baseline neutrino oscillation experiment in Japan. A comparison of the NA61/SHINE measurements with predictions of several hadroproduction models is presented.

Mécanismes de transfert de proton d’une réaction acido-basique en phase aqueuse : une étude ab-initio

Les réactions de transfert de proton se retrouvent abondamment dans la nature et sont des processus cruciaux dans plusieurs réactions chimiques et biologiques, qui se produisent souvent en milieu aqueux. Les mécanismes régissant ces échanges de protons sont complexes et encore mal compris, suscitant un intérêt des chercheurs en vue d’une meilleure compréhension fondamentale du processus de transfert. Le présent manuscrit présente une étude mécanistique portant sur une réaction de transfert de proton entre un acide (phénol fonctionnalisé) et une base (ion carboxylate) en phase aqueuse. Les résultats obtenus sont basés sur un grand nombre de simulations de dynamique moléculaire ab-initio réalisées pour des systèmes de type « donneur-pont-accepteur », où le pont se trouve à être une unique molécule d’eau, permettant ainsi l’élaboration d’un modèle cinétique détaillé pour le système étudié. La voie de transfert principalement observée est un processus ultra-rapide (moins d’une picoseconde) passant par la formation d’une structure de type « Eigen » (H9O4+) pour la molécule d’eau pontante, menant directement à la formation des produits. Une seconde structure de la molécule d’eau pontante est également observée, soit une configuration de type « Zündel » (H5O2+) impliquant l’accepteur de proton (l’ion carboxylate) qui semble agir comme un cul-de-sac pour la réaction de transfert de proton.

Space station and space shuttle programs : hearing before the Subcommittee on Science, Technology, and Space of the Committee on Commerce, Science, and Transportation, United States Senate, One Hundred Fourth Congress, second session, July 24, 1996.

Shipping list no.: 97-0097-P.