Quantitative dissolution of the membrane and preparation of photoreceptor subunits from Rhodospirillum rubrum

Loach, Paul A.; Hadsell, R. M.; Sekura, Diane L.; Stemer, A.

doi:10.1021/bi00818a003

Cited by 39 publications

(13 citation statements)

References 32 publications

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“…This photo-signal has a Gaussian line shape, a g-value of 2.0025, and a linewidth AHpp of about 10 G. Esr investigations with 1H-, 2H-, 13C-, and 21Mg-organisms and their corresponding chlorophylls (2)(3)(4)(5)(6)(7)(8)(9)(10) have demonstrated the ir-nature of the in vivo photo-esr signal. Quantitative and kinetic correlations of the esr and optical spectra have established that the photo-esr signal originates (as originally suspected by Commoner et al) in a chlorophyll (or bacteriochlorophyll) doublet state free radical (Chl at or Bchlt) (11)(12)(13)(14)(15)(16).…”

mentioning

confidence: 75%

An Electron-Nuclear Double Resonance (Endor) Study of the Special Pair Model for Photo-Reactive Chlorophyll in Photosynthesis

Norris

Scheer

Druyan

et al. 1974

Proc. Natl. Acad. Sci. U.S.A.

109

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A comparison of hyperfine coupling constants obtained by electron-nuclear double resonance spectroscopy of in vitro monomer chlorophyll and bacteriochlorophyll free radicals with those of the photoesr (electron spin resonance) signal associated with light conversion in photosynthesis provides convincing support for the special pair model for the in vivo photo-reaction center. The special pair model for photo-active chlorophyll predicts that the esr line shape will be narrowed by a factor 1/A/2 relative to monomer Chl at, and that each and every electronnuclear hyperfine (hf) coupling constant in a special pair will be reduced by a factor of 1/2 relative to the monomer coupling constants. A comparison of hf splittings measured in both in vivo and in vitro chlorophyll free radicals is thus a considerably more rigorous test of the special pair proposal. Because chlorophyll free radical esr signals, especially in vivo, do not show hf structures that permit extraction of hf coupling constants, we have had resort to electron-nuclear double resonance (endor) spectroscopy, a high resolution extension of esr first discovered by G. Feher (22), which in combination with organisms and chlorophylls of unnatural isotopic composition makes assignment of the endor spectrum and hf coupling constants possible. MATERIALS AND METHODSInstrumentation. Endor spectra were recorded at 10'K and 108'K on a modified Varian E-700 spectrometer.Production of Chlorophyll Free Radicals. In vitro free radicals were usually generated in 10-4 10-1 M Chl solutions in thoroughly degassed C2H2C12-C2H302H by chemical oxidation with minimal amounts of iodine (23), ferric chloride, or zinc tetraphenylporphyrin free radical (24).In vivo free radicals for endor were generated in packed whole cells, chromatophores, chloroplasts, and reaction center preparations by careful titration with KFe(CN)6. The esr and endor signals produced by this chemical method are indistinguishable from the photo-induced signal in bacteria (16) and green algae and thus are believed to have the same origin.

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mentioning

confidence: 75%

An Electron-Nuclear Double Resonance (Endor) Study of the Special Pair Model for Photo-Reactive Chlorophyll in Photosynthesis

Norris

Scheer

Druyan

et al. 1974

Proc. Natl. Acad. Sci. U.S.A.

109

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“…They are generally referred to as reaction center prep arations. Another pigment-protein complex which has been extensively studied and characterized (Loach et al, 1970a;1970b) is the so-called 'photoreceptor subunits' isolated from Rhodospirillum rubrum using alkaline, urea and Triton X-100. This unit retains a portion of the antenna pigments.…”

Section: Introductionmentioning

confidence: 99%

“…Literature is cited on an elective rather than a comprehensive basis. For detailed accounts of other 1 1 1 1 1 1 1 aspects of bacterial photosynthesis, the reader is referred to recent reviews (Gest et ul., 1963;Govindjee, 1974;Parson and Cogdell, 1975;Frenkel, 1970;Walker and Crofts, 1970;Clayton, 1973;Vernon, 1968;Loach and Hales, 1976;.…”

Section: Introductionmentioning

confidence: 99%

A Survey of Epr Investigations of Bacterial Photosynthesis

Corker

1976

Photochem & Photobiology

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Abstract-Investigations in which EPR has been used as a probe of the mechanism of the primary quantum conversion reaction and/or electron transport reactions in bacterial photosynthesis are surveyed. These investigations include studies of whole cell organisms and simpler sub-cellular preparations, chromatophores and bacteriochlorophyll-protein complexes.Electron paramagnetic resonance studies have successfully demonstrated that the primary donor of the photosynthetic, photochemical reaction involves a dimer of bacteriochlorophyll, generally referred to as P870. P870 is photochemically oxidized to a cation radical which exhibits a g = 2.0025 EPR signal. Comparative studies of the time behaviour of this signal in whole cells and in sub-cellular preparations show that electron flow in the whole cells is substantially different than in the cell-free systems.The primary acceptor molecule of the photochemical reaction has not been conclusively identified. When it is photochemically reduced, it exhibits a broad EPR absorbance centered at g = 1.8 observable only at low temperatures. This signal involves an iron atom and a quinone molecule. Two possible identifications of the species responsible for the g = 1.8 signal are an iron-quinone complex and an iron-sulfur protein. The latter identification would require that one primary acceptor function for two primary donors and that the removal of a tightly held quinone alter the integrity of the system so as to inhibit the photochemistry.When the primary acceptor is chemically reduced, a photo-induced, polarized triplet EPR spectrum is detected. Both absorption and emission lines arq observed as if only one substate (m = 0) of the triplet manifold is populated. The zero field parameters of the triplet spectrum suggest that the triplet is formed through a decay of a biradical, not through an optical singlet to triplet transition.Low temperature EPR studies of photosynthetic preparations which had been poised at room temperature by subjecting the preparations to different redox potentials and/or dark adaptation and illumination show the presence of a number of light-influenced, EPR active components. The spectral characteristics of these components are indicative of iron-heme (both low and high-spin forms) and iron-sulfur (both oxidized and reduced) proteins and at least one other organic free radical distinct from P870'. The spectra varied for different strains of bacteria. Also, some of the signals detected in the whole cell organism were not detected in cell free preparations and the kinetics of the light influenced signal amplitudes were different in the whole cells than in chromatophores.

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“…The problems inherent in the solubilization of biological membranes, however, have limited the applicability of this method in the fractionation of membrane-bound proteins. In several such studies [2][3][4] membranes solubilized and focused in the presence of 6-8 M urea (and/or detergent) were resolved into a number of protein bands. Under such conditions which promote protein denaturation, however, identification of the proteins is difficult due to lack of suitable assay methods.…”

Section: Introductionmentioning

confidence: 99%

Isoelectric focusing of rod outer segment membrane proteins

1973

FEBS Letters

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Quantitative dissolution of the membrane and preparation of photoreceptor subunits from Rhodospirillum rubrum

Cited by 39 publications

References 32 publications

An Electron-Nuclear Double Resonance (Endor) Study of the Special Pair Model for Photo-Reactive Chlorophyll in Photosynthesis

An Electron-Nuclear Double Resonance (Endor) Study of the Special Pair Model for Photo-Reactive Chlorophyll in Photosynthesis

A Survey of Epr Investigations of Bacterial Photosynthesis

Isoelectric focusing of rod outer segment membrane proteins

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