The bc 1 complexes of Rhodobacter sphaeroides and Rhodobacter capsulatus

Photosynthetic bacteria offer excellent experimental opportunities to explore both the structure and function of the ubiquinol-cytochrome c oxidoreductase ( bc 1 complex). In both Rhodobacter sphaeroides and Rhodobacter capsulatus , the bc 1 complex functions in both the aerobic respiratory chain and as an essential component of the photosynthetic electron transport chain. Because the bc 1 complex in these organisms can be functionally coupled to the photosynthetic reaction center, flash photolysis can be used to study electron flow through the enzyme and to examine the effects of various amino acid substitutions. During the past several years, numerous mutations have been generated in the cytochrome b subunit, in the Rieske iron-sulfur subunit, and in the cytochrome c 1 subunit. Both site-directed and random mutagenesis procedures have been utilized. Studies of these mutations have identified amino acid residues that are metal ligands, as well as those residues that are at or near either the quinol oxidase (Q o ) site or the quinol reductase (Q i ) site. The postulate that these two Q-sites are located on opposite sides of the membrane is supported by these studies. Current research is directed at exploring the details of the catalytic mechanism, the nature of the subunit interactions, and the assembly of this enzyme.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44795/1/10863_2004_Article_BF00762582.pd

Interruption of the Water Chain in the Reaction Center from Rhodobacter sphaeroides Reduces the Rates of the Proton Uptake and of the Second Electron Transfer to Q_B

A chain of bound water molecules was recently identified in the photosynthetic reaction center (RC) from Rhodobacter sphaeroides by X-ray crystallography [Ermler et al. (1994) Structure 2, 925-936]. The possible role of the chain in proton transfer from the solution to the secondary quinone (QB) was investigated by site-directed mutagenesis and flash-induced absorbance spectroscopy. Pro L209, situated along the water chain about 9 A from QB, was changed into the aromatic residues Phe and Tyr in order to interrupt the chain. In the PL209Y (Pro L209-->Tyr) mutant, the very small changes in the QA-QB QAQB- equilibrium constant (K2) and the first electron-transfer rates (kAB(1)) indicate that the mutation does not lead to large structural changes. In the PL209F (Pro L209-->Phe) mutant, a 7-fold decrease of kAB(1) is observed. It follows a pH dependence parallel to that of the wild type. It is consistent with no modification of the pK of the Glu L212 determined from the pH dependence of K2. The decreased kAB(1) may reflect some slight structural modification in this mutant and/or rearrangement of the cluster of charged residues close to the L209 position. The major effect of the mutations observed is a concomitant decrease of the rates of the second electron transfer, kAB(2), and of the proton uptake upon the second flash. The relative decrease of the kAB(2) rate values in the mutants is more pronounced above pH 8. Our results indicate that the mutations have specifically altered the pathway of proton transfer to QB</sub

Study of wild type and genetically modified reaction centers from Rhodobacter capsulatus: structural comparison with Rhodopseudomonas viridis and Rhodobacter sphaeroides.

Reaction centers from the purple bacterium Rhodobacter (Rb.) capsulatus and from two mutants ThrL226-->Ala and IleL229-->Ser, modified in the binding protein pocket of the secondary quinone acceptor (QB), have been studied by flash-induced absorbance spectroscopy. In ThrL226-->Ala, the binding affinities for endogenous QB (ubiquinone 10) and UQ6 are found to be two to three times as high as the wild type. In contrast, in IleL229-->Ser, the binding affinity for UQ6 is decreased about three times compared to the wild type. In ThrL226-->Ala, a markedly increased sensitivity (approximately 30 times) to o-phenanthroline is observed. In Rhodopseudomonas viridis, where Ala is naturally in position L226, the sensitivity to o-phenanthroline is close to that observed in ThrL226-->Ala. We propose that the presence of Ala in position L226 is responsible for the high sensitivity to that inhibitor. The pH dependencies of the rate constants of P+QB- (kBP) charge recombination kinetics (P is a dimer of bacteriochlorophyll, and QB is the secondary quinone electron acceptor) show destabilization of QB- in ThrL226-->Ala and IleL229-->Ser, compared to the wild type. At low pH, similar apparent pK values of protonation of amino acids around QB- are measured in the wild type and the mutants. In contrast to Rb. sphaeroides, in the wild type Rb. capsulatus, kBP substantially increases in the pH range 7-10. This may reflect some differences in the respective structures of both strains or, alternatively, may be due to deprotonation of TyrL 215 in Rb. capsulatus. At pH 7, measurements of the rate constant of QA to QB electron transfer reveal a threefold greater rate in the reaction centers from wild type Rb. capsulatus (65 +/- 1 0 ps)-1 compared to Rb. sphaeroides.We suggest that this may arise from a 0.7-A smaller distance between the quinones in the former strain. Our spectroscopic data on the wild type Rb. capsulatus reaction center suggest the existence of notable differences with the Rb. sphaeroides reaction center structure

In Rhodobacter sphaeroides Reaction Centers, Mutation of Proline L209 to Aromatic Residues in the Vicinity of a Water Channel Alters the Dynamic Coupling between Electron and Proton Transfer Processes

The X-ray crystallographic structure of the photosynthetic reaction center from Rhodobactersphaeroides obtained at high resolution has revealed a number of internal water molecules (Ermler, U., Fritzsch, G., Buchanan, S. K., and Michel, H. (1994) Structure2, 925−936; Stowell, M. H. B., McPhillips, T. M., Rees, D. C., Soltis, S. M., Abresch, E., and Feher, G. (1997) Science276, 812−816). Some of them are organized into distinct hydrogen-bonded water chains that connect QB (the terminal quinone electron acceptor of the reaction center) to the aqueous phase. To investigate the role of the water chains in the proton conduction process, proline L209, located immediately adjacent to a water chain, was mutated to the following residues:  F, Y, W, E, and T. We have first analyzed the effects of the mutations on the kinetic and thermodynamic properties of the rate constants of the second electron transfer (kAB(2)) and of the coupled proton uptake (kH+) at the second flash. In all aromatic mutants, kAB(2) and kH+ are notably and concomitantly decreased compared to the wild-type, while no effect is observed in the other mutants. The temperature dependence of these rates shows activation energy values (ΔH⧧) similar for the proton and electron-transfer processes in the wild-type and in most of the mutants, except for the L209PW and L209PF mutants. The analysis of the enthalpy factors related to the electron and proton-transfer processes in the L209PF and the L209PW mutants allows to distinguish the respective effects of the mutations for both transfer reactions. It is noteworthy that in the aromatic mutants a substantial increase of the free energies of activation is observed (ΔG⧧L209PY ⧧L209PF ⧧L209PW) for both proton and electron-transfer reactions, while in the other mutants, ΔG⧧ is not affected. The salt concentration dependence of kAB(2) shows, in the L209PF and L209PW mutants, a higher screening of the protein surface potential experienced by QB. Our data suggest that residues F and W in position L209 increase the polarizability of the internal water molecules and polar residues by altering the organization of the hydrogen-bond network. We have also analyzed the rates of the first electron-transfer reaction (kAB(1)), in the 100 μs time domain. These kinetics have previously been shown to reflect protein relaxation events possibly including proton uptake events (Tiede, D. M., Vazquez, J., Cordova, J., and Marone, P. M. (1996) Biochemistry 35, 10763−10775). Interestingly, in the L209PF and L209PW mutants, kAB(1) is notably decreased in comparison to the wild type and the other mutants, in a similar way as kAB(2) and kH+. Our data imply that the dynamic organization of this web is tightly coupled to the electron transfer process that is kinetically limited by protonation events and/or conformational rearrangements within the protein

Etat des lieux de l'utilisation de la Kétamine par les SAMU-SMUR de Lorraine

[Résumé en français] Nous avons réalisé une étude portant sur l'utilisation de la kétamine en préhospitalier en Lorraine. La kétamine est une molécule originale pour l'anesthésie dissociative qu'elle procure, son action sympathomimétique et son action bronchodilatatrice. Elle est adaptée à la médecine préhospitalière. Notre objectif est de dresser un état des lieux de son utilisation en Lorraine en préhospitalier et d'évaluer le respect des indications et des posologies proposées dans la littérature. Pour cela, nous avons réalisé une enquête en adressant un questionnaires à l'ensemble des médecins lorrains pratiquant la médecine préhospitalière. Il ressort de notre étude que les médecins lorrains utilisent fréquemment cette molécule. Les indications cliniques retrouvées dans la littérature sont respectées. Le point principal de notre travail est la mise en évidence d'une grande disparité des posologies administrées. Cette disparité est principalement due à l'absence de protocole dans certains services et à l'absence de leur diffusion à l'équipe médicale dans d'autres services. Les nombreux effets secondaires décrits par les médecins interrogés sont principalement dus à une mauvaise utilisation de la molécule (doses et administration inadaptées, association au midazolam et/ou à l'atropine non systématique). Dans l'avenir l'établissement d'un protocole dans chaque service et la diffusion à l'ensemble des médecins permettra d'améliorer et d'harmoniser l'utilisation de la kétamine en Lorraine. De même les effets secondaires constatés seront diminués lors de la mise sur le marché français de la forme dextrogyre.[Résumé en anglais] We have carried out a study on the use of the ketamine in the prehospital sector in Lorraine. The ketamine is original as a molecule due to its dissociative anesthetic action, its sympathomimetic action and its bronchodilatating action. It is adapted to the prehospital medicine. Our objective with this study is to estimate how widely it is used in the prehospital sector in Lorraine and to evaluate if the directions for use and dosage that are found in the literature are respected in practice. To do so, we have carried out a survey by sending out a questionnaire to ail practicing doctors in the prehospital sector who work in Lorraine. The conclusion of the survey is that, in the Lorraine region, doctors often use this molecule. We also find that the clinical directions given in the literature for its use are respected in practice. The key element of our study is to highlight the fact that there exists a huge disparity in the given dosage. This disparity is mainly due to the lack of the existence of a protocol in sorne services or, to the lack of the communication of this protocol to the medical team in other services. The many side effects described by the surveyed doctors are mainly due to the wrong uses of the molecule. (These include for example unsuitable amounts being used or administered or the systematic failure to associate sorne midazolam and/or atropine with it). ln the future, establishing a protocol in every service and its diffusion to all doctors will allow improving and harmonizing the use of the ketamine in Lorraine. This will aIso allow for a diminution of the observed side effects when the dextrogyre is launched on the French market.NANCY1-SCD Medecine (545472101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

X-ray Structure Analyses of Photosynthetic Reaction Center Variants from Rhodobacter sphaeroides: Structural Changes Induced by Point Mutations at Position L209 Modulate Electron and Proton Transfer

The structures of the reaction center variants Pro L209 → Tyr, Pro L209 → Phe, and Pro L209 → Glu from the photosynthetic purple bacterium Rhodobacter sphaeroides have been determined by X-ray crystallography to 2.6−2.8 Å resolution. These variants were constructed to interrupt a chain of tightly bound water molecules that was assumed to facilitate proton transfer from the cytoplasm to the secondary quinone QB [Baciou, L., and Michel, H. (1995) Biochemistry34, 7967−7972]. However, the amino acid exchanges Pro L209 → Tyr and Pro L209 → Phe do not interrupt the water chain. Both aromatic side chains are oriented away from this water chain and interact with three surrounding polar side chains (Asp L213, Thr L226, and Glu H173) which are displaced by up to 2.6 Å. The conformational changes induced by the bulky aromatic rings of Tyr L209 and Phe L209 lead to unexpected displacements of QB compared to the wild-type protein. In the structure of the Pro L209 → Tyr variant, QB is shifted by ∼4 Å and is now located at a position similar to that reported for the wild-type reaction center after illumination [Stowell, M. H. B., et al. (1997) Science276, 812−816]. In the Pro L209 → Phe variant, the electron density map reveals an intermediate QB position between the binding sites of the wild-type protein in the dark and the Pro L209 → Tyr protein. In the Pro L209 → Glu reaction center, the carboxylic side chain of Glu L209 is located within the water chain, and the binding site of QB remains unchanged compared to the wild-type structure