Rhodobacter sphaeroides methods

P. Beroza, D. R. Fredkin, M. Y. Okamura, and G. Feher, Proc. Natl. Acad. Sci. U.S.A., 88, 5804 (1991). Protonation of Interacting Residues in a Protein by a Monte Carlo Method Application to Lysozyme and the Photosynthetic Reaction Center of Rhodobacter sphaeroides. 

Beroza, R, Fredkin, D.R., Okamura, M.Y., Feher, G. Protonation of interacting residues in a protein by a Monte Carlo method Application to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides. Proc. Natl. Acad. Sci. USA 1991, 88, 5804-8. 

Fig. 1. The three different interaction ranges ofEPR spectroscopy are shown on a transient semiquinone anion radical QX of a bacterial reaction centre of Rhodobacter sphaeroides (R26). The structural data for ttiis illustration are taken from the X-ray structure of Ermler et al. [2]. Upper left The local interactions such as intermolecular hyperfine couplings and g-tensor of the ubisemiquinone-10 anion radical characterized by multifrequency-EPR. Upper right The local protein surrounding (within a distance of 0.6 nm to the semiquinone rachcal) as observable by ENDOR and ESEEM spectroscopies. Lower picture The interaction of the semiquinone anion radical with the other transient paramagnetic centres in the protein complex as observable with pulse-EPR methods.

The three dimensional structure of the reaction center (RC) from Rhodobacter sphaeroides R-26 and 2.4.1 has been reported at a resolution of 2.8 A and 3.0 A respectively (1-4). To improve the accuracy of these models we have futher refined the R-26 data using molecular dynamics methods. We have also collected diffraction data at higher resolution for RCs from the 2.4.1 strain. To relate the three dimensional structure to its function, we are performing various studies on RCs with modified structures (altered amino acid composition, cofactors removed, with herbicide bound). We describe the structure of RCs containing only the primary quinone and the structure of RCs with the herbicide, terbutryn, bound. Progress in crystallizing and determining the structures of other modified RCs is also reported. 

X-ray diffraction methods have provided the detailed structures of the reaction centers from two carotenoid-containing puiple photosynthetic bacterial species, Rhodopseudomonas viridis [1] and Rhodobacter sphaeroides wild type strain 2.4.1 [2]. The coordinates of these structures indicate that the reaction center-bound carotenoid is located in the M subunit, close ( 4A) to the accessory bacteriochlorophyll monomer on the M subunit side and -lO.SA edge-to-edge distance from the primary donor. These structures suggest an involvement of the M-side monomeric bacteriochlorophyll in triplet-triplet energy transfer, but there has been no direct experimental verification of this hypothesis. 

Rhodobacter sphaeroides WS8 (wild type) was grown, as previously described [6], at 25 C under constant illumination. Cells were harvested and resuspended in either lOmM Hepes-NaOH (pH7.2) or, in the case of cells intended for the preparation of chromatophores, in 50mM NaCl, 8mM MgCl2f 290mM sucrose, 50mM Tricine-NaOH (pH7.4). Chromatophores were prepared by the method described by Jackson and Nicholls [7]. ... 

Although they have been presented before, we review for comparison and a new perspective the methods used to simulate the polarized spectrum p(-band, protonated) of the P Q radical pair of Rhodobacter sphaeroides. The evolution of the density matrix for both the CRPP and the CIDEP treatments is described by... 

RCs from Rhodobacter sphaeroides R-26 were purified with the solubilizing detergent LDAO as described (16). RCs with one quinone, i.e. Q, were prepared by the method of Okamura et al. (17). Cytochrome c (horse heart type VI, Sigma, MO) was more than 95% reduced with hydrogen gas, using platinum black (Alchich, WI) as a catalyst (18). Soybean lecithin (Sigma, MO) was purified as described in (19). 

The crystallization of RCs in view of structure determination by x-ray crj taUography requires special methods that have worked successfully for two kinds of purple bacteria Rhodopseudomonas viridis and Rhodobacter sphaeroides) and for Photosystems I and II of thermophilic cyanobacteria. 

It has been known for some time (see c.g. Ref. 2) that illuminating RCs of Rhodobacter (Rb.) sphaeroides at room temperature in the presence of an extraneous electron donor in a medium poised at low redox potential, leads to the stable photorcduction of the acceptor BPhcL. Five years ago, we showed that when methylviologcn (MV) was used as an extraneous electron donor, illumination at room temperature of the RCs lead to the selective photorcduction of the usually inactive BPhe, namely BPhcM (3). Although the mechanisms responsible for this reaction were not totally clear, this method docs constitute a powerful tool in studying the degree of symmetry existing between the two branches of the bacterial RC. In this paper, we report the effect of the reduction of either of the BPhe molecules on the low temperature absorption spectra of Rb. sphaeroides RC. These results provide strong... 

Electron transfer within the bacterial photosynthetic reaction center (RC) has been shown by a variety of methods to only occur along one branch of the chromophores (the so-called L or A branch) (Kirmaier and Holten, 1987 Parson, 1987). Recent room temperature kinetic data support a model in which the electronically excited primary donor (a dimer of bacteriochlorophylls, P) decays with a time constant of 3.5 ps (in Rhodobacter (Rb,) sphaeroides) to populate a very short-lived intermediate containing P+ and a reduced bacteriochlorophyll (B") molecule (Holzapfel et al, 1989,1990). Subsequently the electron is transferred to a bacteriopheophytin (H) with a time constant of 0.9 ps. The existence of the state P+H is generally accepted but there is presently debate over the existence of the... 

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