A rabbit antiserum was raised against the photoactive yellow protein (PYP) from Ectothiorhodospira halophila and purified by adsorption experiments to obtain a highly specific polyclonal antiserum. This antiserum was used to obtain the following results. (i) In E. halophila, PYP can be isolated from the fraction of soluble proteins. In the intact cell, however, PYP appeared to be associated with (intra)cytoplasmic membranes, as was concluded from analysis of immunogold-labelled thin sections of the organism. (ii) The regulation of expression of PYP was studied by using dot blot assays, Western blotting (immunoblotting), and rocket immunoelectrophoresis. Under all conditions investigated (light color, salt concentration, and growth phase), PYP was expressed constitutively in E. halophila. However, when Rhodospirillum salexigens was grown aerobically, the expression of PYP was suppressed. (iii) A large number of prokaryotic microorganisms contained a single protein, with an apparent size of approximately 15 kDa, that cross-reacted with the antiserum. Among the positively reacting organisms were both phototrophic and chemotrophic, as well as motile and nonmotile, organisms. After separation of cellular proteins into a membrane fraction and soluble proteins, it was established that organisms adapted to growth at higher salt concentrations tended to have the cross-reacting protein in the soluble fraction. In the cases of R. salegens and Chromatium salexigens, we have shown that the cross-reacting protein involved is strongly homologous to PYP from E. halophila.Membrane-bound photoactive proteins are known to function in Halobacterium halobium. In this organism and in related bacteria, retinal-containing proteins have a role in light energy transduction and in phototactic responses (for reviews, see references 29 and 31). A few years ago, the presence of a water-soluble protein of 14 kDa with a distinct yellow color was described in the purple sulfur eubacterium Ectothiorhodospira halophila (15). This protein is photoactive: it displays a photocycle that is quite similar to the photocycle of the sensory rhodopsins (10,20,22,23). Its three-dimensional structure, which is unrelated to the structure of the bacterial rhodopsins, has been elucidated at 2.4-A (0.24-nm) resolution (14). It consists of two perpendicular plates of A sheet, forming a ,3-clam structure, also observed in a number of eukaryotic proteins. Evidence indicating that the protein functions as the photoreceptor in a negative phototactic response has been obtained (28). Despite the photochemical and functional similarities between the photoactive yellow protein (PYP) and the sensory rhodopsins, the chromophoric group in PYP is not retinal (33).Recently, PYPs were purified from Rhodospirillum salexigens (19) and Chromatium salexigens (18). Therefore, PYP is now known to be present in halophilic representatives of three * Corresponding author. Mailing address:

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Ubiquinol-cytochrome c oxidoreductase (cytochrome bej) complexes were demonstrated to be present in the membranes of the alkaliphilic and halophilic purple sulfur bacteria Ectothiorhodospira halophila, Ectothiorhodospira mobilis, and Ectothiorhodospira shaposhnikovii by protoheme extraction, immunoblotting, and electron paramagnetic resonance spectroscopy. The gy values of the Rieske [2Fe-2S] clusters observed in membranes of E. mobifis and E. halophila were 1.895 and 1.910, respectively. In E. mobilis membranes, the cytochrome be1 complex was present in a stoichiometry of approximately 0.2 per reaction center. This complex was isolated and characterized. It contained four prosthetic groups: low-potential cytochrome b (cytochrome bL; Em = -142 mV), high-potential cytochrome b (cytochrome bH; E. = 116 mV), cytochrome cl (Em = 341 mV), and a Rieske iron-sulfur cluster. The absorbance spectrum of cytochrome bL displayed an asymmetric a-band with a maximum at 564 nm and a shoulder at 559 nm. The a bands of cytochrome bH and cytochrome cl peaked at 559.5 and 553 nm, respectively. These prosthetic groups were associated with three different polypeptides: cytochrome b, cytochrome cl, and the Rieske iron-sulfur protein, with apparent molecular masses of 43, 30, and 21 kDa, respectively. No evidence for the presence of a fourth subunit was obtained.Maximal ubiquinol-cytochrome c oxidoreductase activity of the purified complex was observed at pH 8; the turnover rate was 57 mol of cytochrome c reduced (mol of cytochrome cj)-1 s-1. The complex showed a strikingly low sensitivity towards typical inhibitors of cytochrome bc complexes.

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Properties of the primary and secondary quinone electron acceptors in RC/LH1 complexes from the purple sulfur bacterium Ectothiorhodospira mobilis. Leguijt, T.; Parot, P.; Vermeglio, A.; Crielaard, W.; Hellingwerf, K.J. Published in:Biochimica et Biophysica Acta G General Subjects Link to publication Citation for published version (APA):Leguijt, T., Parot, P., Vermeglio, A., Crielaard, W., & Hellingwerf, K. J. (1994). Properties of the primary and secondary quinone electron acceptors in RC/LH1 complexes from the purple sulfur bacterium Ectothiorhodospira mobilis. Biochimica et Biophysica Acta G General Subjects, 1183, 292-300. General rightsIt is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), other than for strictly personal, individual use, unless the work is under an open content license (like Creative Commons). Disclaimer/Complaints regulationsIf you believe that digital publication of certain material infringes any of your rights or (privacy) interests, please let the Library know, stating your reasons. In case of a legitimate complaint, the Library will make the material inaccessible and/or remove it from the website. Please Ask the Library: http://uba.uva.nl/en/contact, or a letter to: Library of the University of Amsterdam, Secretariat, Singel 425, 1012 WP Amsterdam, The Netherlands. You will be contacted as soon as possible. Reaction centers (RCs)with one antenna complex attached (RC/LH~ complexes)were isolated from the alkaliphilic and moderately halophilic, phototrophic purple sulfur bacterium Ectothiorhodospira (E.) mobilis and analyzed with respect to quinone composition and charge recombination kinetics, using time-resolved (low-and room-temperature) spectroscopy and thin-layer chromatography. The RCs contain menaquinone as primary and ubiquinone as secondary quinone electron acceptor, QA and Q a, respectively. Q B is lost during isolation of the RC/LH I complexes. P+ QA charge recombination kinetics, which were shown to be pH independent, were only slightly temperature dependent, indicating that this recombination proceeds via the direct (electron tunnelling) route. At room temperature, its average lifetime was 34.5 ms. These decay kinetics were shown to be monophasic at room temperature and biphasic at low temperature. Addition of an excess of UQ 6 to RC/LH I complexes resulted in retardation of the P+ recovery, due to charge recombination of the state P+QAQa. Functional reconstitution of QB was also evident from flash-induced binary oscillations at 450 nm, which could be observed in RC/LH I complexes in the presence of an excess of UQ 6. This reconstitution of QB activity was, however, incomplete and decreased with increasing pH. The P+QAQB decay kinetics were pH independent; the average lifetime was about 6 s at room temperature. The apparent equilibrium constant g 2 between the states QAQB and QAQB, and consequently the free energy difference between these states, were relatively large and pH independent...