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R K Clayton

Publications and source records attributed to R K Clayton.

At least 19 recordsLinked to original sources
PubMed1981

B850 pigment-protein complex of Rhodopseudomonas sphaeroides: Extinction coefficients, circular dichroism, and the reversible binding of bacteriochlorophyll.

Chromatophores of Rhodopseudomonas sphaeroides yield the antenna complex B850 in either of two states, depending on the method of isolation. Methods using dodecyl (= lauryl) dimethylamine oxide yield B850 with an absorption spectrum like that in vivo: the bands at 800 and 850 nm, due to the bacteriochlorophyll (Bchl) components Bchl-800 and Bchl-850, are in ratio A(800)/A(850) = 0.65 +/- 0.05. When B850 is isolated by methods using dodecyl sulfate, the Bchl-800 is attenuated or absent. Bchl assays of these materials and of the isolated antenna complex B875 yielded the following extinction coefficients, +/-SD, on the basis of the molarity of Bchl: For B875, epsilon(875) = 126 +/- 8 mM(-1) cm(-1). For B850 in the normal (high-Bchl-800) state, epsilon(850) = 132 +/- 10 mM(-1) cm(-1). For the individual components of Bchl in B850, epsilon(850) of Bchl-850 = 184 +/- 13 mM(-1) cm(-1) and epsilon(800) of Bchl-800 = 213 +/- 28 mM(-1) cm(-1). With these coefficients the molecular ratio of Bchl-850 to Bchl-800 equals 1.8 +/- 0.4 for B850 in the high-Bchl-800 state. Starting with B850 depleted of Bchl-800, the addition of dodecyldimethylamine oxide restored the 800-nm absorption band. The 850-nm band became shifted toward the blue, narrowed, and slightly attenuated, and its associated circular dichroism became 20% more intense. Free Bchl added with dodecyldimethylamine oxide accelerated the restoration of Bchl-800 and retarded the attenuation of Bchl-850. We conclude that free Bchl can interact reversibly with a binding site for Bchl-800 in the B850 complex, with dodecyl sulfate favoring dissociation and dodecyldimethylamine oxide promoting association. Thus the reversible dissociation of a native chlorophyll-protein complex has now been demonstrated.

Journal Article↗
PubMed1980

Orientation of the hemes of high potential cytochromes relative to photosynthetic membranes, as shown by the linear dichroism of oriented preparations.

The orientations of high potential cytochromes with respect to photosynthetic membranes was investigated in spinach chloroplasts and in Rhodopseudomonas viridis. The general approach consists in detection with polarized light of photoinduced absorbance changes related to the oxidation of the cytochromes. The orientation of cytochrome c-558 was measured at room temperature in chromatophores and whole cells of Rps. viridis, oriented on glass slides and in a magnetic field, respectively. The orientation of cytochrome b-559 of green plants was detected at 77 K in magnetically oriented chloroplasts. In both cases the dichroic ratio for the alpha band shows that the heme plane makes an angle greater than 35 degrees C with the membrane plane. Moreover, the dichroic ratio is not constant throughout the alpha and beta bands, for both cytochrome c-558 and b-559. Linear dichroism spectra of oriented pure horse heart cytochrome c and cytochrome c2 of Rhodopseudomonas sphaeroides in stretched polyvinyl alcohol films show that the variations of the dichroic ratio in the alpha and beta bands can be explained by the occurrence of x- and y-polarized transitions absorbing at slightly different wavelengths.

Chloroplasts↗
PubMed1980

Isolation and characterization of the pigment-protein complexes of Rhodopseudomonas sphaeroides by lithium dodecyl sulfate/polyacrylamide gel electrophoresis.

When purified photosynthetic membranes from Rhodopseudomonas sphaeroides were treated with lithium dodecyl sulfate and subjected to polyacrylamide gel electrophoresis at 4 degrees C, up to 11 pigment-protein complexes were resolved. Absorption spectra revealed that the smallest complex contained reaction center pigments and the others contained the antenna components B850 and B875 in various proportions. Of these antenna complexes, the largest was almost entirely B850 and the smallest contained only B875. After solubilization at 100 degrees C and electrophoresis on polyacrylamide gradient gels, the B850 complex gave rise to two polypeptide components migrating with apparent Mr of 10,000 and 8000, whereas with the B875 complex, two components were observed with apparent Mr of 12,000 and 8000. The reaction center complex gave rise to only the 24 and 21 kilodalton polypeptide subunits. Fluorescence emission spectra showed maxima at 872 and 902 nm for B850 and B875, respectively. Analyses of bacteriochlorophyll a and carotenoids indicated that, in the B875 complex, two molecules of each of these pigments are associated with the two polypeptides. The associations of B850 and B875 in large and small complexes obtained by lithium dodecyl sulfate treatment are consistent with models of their organization within the membrane.

Bacteriochlorophylls↗
PubMed1980

Mode of inhibition of electron transport by orthophenanthroline in chromatophores and reaction centers of Rhodopseudomonas sphaeroides.

In Rhodopseudomonas sphaeroides, light causes the transfer of electrons from bacteriochlorophyll to ubiquinone in the photochemical reaction centers. Electrons from this "primary" ubiquinone move on to a secondary ubiquinone. We have studied the manner in which o-phenanthroline inhibits the transfer of electrons from primary to secondary ubiquinone in chromatophores (intracytoplasmic membrane fragments) and isolated reaction centers of Rp. sphaeroides. The formation of anionic semiquinones, both primary and secondary, is signaled by an absorption band at 450 nm and by band shifts of bacteriopheophytin and bacteriochlorophyll in the near infrared. The pattern of band shifts is different for primary and secondary semiquinone, allowing us to distinguish which quinone has become semireduced. This point was established for isolated reaction centers [Vermeglio, A. & Clayton, R. K. (1977) Biochim. Biophys. Acta 461, 159-165] and is confirmed here for chromatophores. By adding o-phenanthroline at various times during a sequence of actinic light flashes and monitoring the resulting optical absorbance changes, we have found that o-phenanthroline inhibits electron transfer from primary semiquinone to secondary quinone if the latter is in its oxidized form, but not if the latter is semireduced. Our findings can explain the decay kinetics of delayed fluorescence from bacteriochlorophyll in Rp. sphaeroides as measured by R. P. Carithers and W. W. Parson [(1976) Biochim. Biophys. Acta 440, 215-232].

Journal Article↗
PubMed1979

The orientations of reaction center transition moments in the chromatophore membrane of Rhodopseudomonas sphareroides, bases on new linear dichroism and photoselection measurements.

Chromatophore membranes from Rhodopseudomonas sphaeroides were oriented by drying suspensions on the surfaces of glass slides, Polarized spectra of light-induced absorption changes were obtained between 500 and 1000 nm. As observed earlier, these spectra showed negative bands, reflecting photooxidation of the bacteriochlorophyll 'special pair' in the reaction centers, centered near 870, 810, 630 and 600 nm. These bands have been designated BY1, BY2, BX1 and BX2, respectively, corresponding to two QY transitions and two QX transitions of the dimeric special pair. We found the BY1 and BX1 transition moments to be parallel (within 20 degrees) to the plane of the membrane, whereas the BX2 moment makes an angle of 55--63 degrees with the plane. Using the photoselection technique we found that the angle between the BY1 and BX1 transition moments is 30 degrees, while that between BY1 and BX2 is 75 degrees. The BX1 and BX2 moments were found to be orthogonal, consistent with the prediction of molecular exciton theory for a dimer. By combining these data, we have calculated the orientations of the transition moments of the bacteriochlorophyll dimer in spherical polar coordinates, with the pole of the coordinate system normal to the plane of the membrane. The orientations of the QY and QX transition moments of the two bacteriopheophytin molecules in the reaction center were also computed in this coordinate system by transforming the data reported by Clayton, C.N., Rafferty, R.K. and Vermeglio, A. ((1979) Biochim. Biophys. Acta 545, 58--68). We have derived the transformation equations for two polar coordinate systems: in one, the pole is an axis of symmetry as defined by the orientations of purified reaction centers in stretched gelatin films (Rafferty, C.N. and Clayton, R.K. (1979) Biochim. Biophys. Acta 545, 106--121). In the other, the pole is normal to the plane of the chromatophore membrane. These two polar axes are approximately orthogonal.

Bacterial Chromatophores↗
PubMed1979

The orientations of transition moments in reaction centers of Rhodopseudomonas sphaeroides, computed from data of linear dichroism and photoselection measurements.

Linear dichroism measurements of reaction centers of Rhodopseudomonas sphaeroides in stretched gelatin films have yielded angles that various optical transition moments make with an axis of symmetry in the reaction center. Photoselection experiments have yielded angles that certain transition moments make with each other. We have combined these data so as to compute the orientations of the Qx and Qy transition moments of the two molecules of bacteriopheophytin and of the bacteriochlorophyll special pair (photochemical electron donor) in the reaction center. Orientations are expressed in spherical polar coordinates with the symmetry axis as the pole. We have also computed additional angles between pairs of transition moments. In this treatment we have assumed that the bacteriopheophytins are independent monomers with little or no exciton coupling.

Bacteriochlorophylls↗
PubMed1979

Linear dichroism and the orientation of reaction centers of Rhodopseudomonas sphaeroides in dried gelatin films.

Aqueous mixtures of reaction centers of Rhodopseudomonas sphaeroides and gelatin were dried to form thin films. Following hydration, these films were stretched as much as two to three times their original length. Polarized absorption spectra showing linear dichroism were obtained for both unstretched and stretched films, with the planes and stretching axes of the films mounted in various geometries relative to the electric vector of the measuring beam. These data were analyzed in terms of the following model: Reaction centers possess an axis of symmetry that is fixed in relation to the reaction center structure. In unstretched films this axis is confined to the film plane and oriented at random within the plane. In stretched films the symmetry axis is aligned with the direction of stretching. In both preparations reaction centers are distributed randomly with respect to rotation about the axis of symmetry. The data are consistent with this model when the analysis acknowledges less than perfect orientation. For perfect orientation in a stretched film the model predicts uniaxial symmetry about the axis of stretching. The approach to this condition was examined with films stretched to different extents. Extrapolation yielded dichroic ratios for the ideal case of perfect orientation, and allowed calculation of the angles between the axis of symmetry and the various optical transition dipoles in the reaction center. This treatment included the two absorption bands of the bacteriochlorophyll 'special pair' (photochemical electron donor) in the Qx region, at 600 and 630 nm, which we were able to resolve in light minus dark difference spectra.

Darkness↗
PubMed1979

Photoreversible absorbance changes in solutions of allophycocyanin purified from Fremyella diplosiphon: Temperature dependence and quantum efficiency.

Preparations of allophycocyanin isolated from the alga Fremyella diplosiphon show light-induced optical absorbance changes that suggest the presence of a photoconvertible component [Formula: see text] similar to the algal pigments described by J. Scheibe [(1972) Science 176, 1037-1039]. At pH < 4 the allophycocyanin has an absorption maximum at 620 nm. Red illumination causes a loss of absorbance in the red, centered at 620 nm, and subsequent green illumination restores the lost absorbance. We have studied this photoconversion at temperatures between 200 K and 307 K, analyzing the results in terms of photostationary states established under red (640 nm) and green (550 nm) light. As the temperature was lowered to 260 K, the state P(r) became progressively favored; the reaction P(r) --> P(g) induced by red light was attenuated but the reaction P(g) --> P(r) induced by green light was not. Decreasing the temperature from 260 K to 200 K had no further effect. Two distinct and simple models can account for this curious temperature dependence. By analyzing the kinetic and steady-state data, with reasonable estimates of the molar extinction coefficients of P(r) and P(g), we computed quantum efficiencies greater than 15% for the photoconversion at 300 K. We deduced that a conversion of "all P(r)" to "all P(g)" should produce a fractional absorbance change DeltaA/A at 620 nm equal to 0.1. If the chromatic adaptation response of intact F. diplosiphon shows the unusual temperature dependence reported here, the system P(r) right harpoon over left harpoon P(g) will be implicated in mediating this response.

Journal Article↗
PubMed1979

Photooxidation of antenna bacteriochlorophyll in chromatophores from carotenoidless mutant Rhodopseudomonas sphaeroides and the attendant loss of dimeric exciton interaction.

Intense continuous illumination of purified chromatophores from carotenoidless mutant Rhodopseudomonas sphaeroides results in progressive photooxidative loss of the near infrared absorption band near 860 nm assigned to antenna bacteriochlorophyll. The quantum yield of this reaction is low, approximately 1.7 x 10(-5). The loss in near infrared absorption is accompanied by a proportional shift in the absorption maximum to shorter wavelengths. The double circular dichroism feature in the near infrared decreases at a faster rate than does the absorbance. These results are explained by a model in which the antenna bacteriochlorophyll, initially associated as dimers (lambda(max) = 860.2 nm), is progressively converted to the monomeric state (lambda(max) = 851.9 nm). The wavelength shift is attributed to disruption of exciton coupling in the dimer. Acetone/methanol extraction indicates that the maximum molar extinction coefficients of the dimer and monomer do not differ by more than 4%. The occurrence of an absorption maximum at 852 nm for monomeric bacteriochlorophyll in a protein complex demonstrates that it is not necessary to invoke aggregation of the chromophores as the origin of the shift from 770 nm in typical organic solvents.

Journal Article↗
PubMed1978

Effects of dehydration on reaction centers from Rhodopseudomonas sphaeroides.

Air-dried films of reaction centers from Rhodopseudomonas sphaeroides were found to resemble aqueous suspensions of these reaction centers in their optical and photochemical properties, except that the long wave absorption band of bacteriochlorophyll was shifted from about 860 to 845 nm. The quantum efficiency of the photochemical reaction that produces oxidized bacteriochlorophyll and reduced ubiquinone was greater than 0.75 in the films, using 800-nm actinic light. Dehydration of the films caused further blue shifts of all the absorption bands between 500 and 900 nm, and some loss (about 20%) of intensity of the long wave band, coupled to a gain of absorbance centered at 795 nm. The latter changes were not accompanied by an increase at 1250 nm, ruling out the oxidation of bacteriochlorophyll as a cause of these changes. Dehydration caused about 50% of the reaction centers to become photochemically inactive. For the component that remained active, the photochemical quantum efficiency was about 0.5 at room temperature, rising to 0.7 or more as the temperature was brought below 250 K. The yield of 900 nm fluorescence was approximately the same in air-dried films as in aqueous suspensions; dehydration of a film raised the fluorescence yield about 3-fold. The yield of this fluorescence in a dehydrated film was independent of temperature (+/- 10%) between 300 and 70 K. The back reaction after illumination, return of electrons from reduced ubiquinone to oxidized bacteriochlorophyll, has kinetics in which a most rapid first-order component is mixed with slower components. The fastest component, identified with the return of electrons from 'primary' ubiquinone to oxidized bacteriochlorophyll, was predominant in the materials tested here. Its half time in an aqueous suspension of reaction centers fell from 86 ms at 300 K to 33 MS AT 200 K and declined more gradually to 15 ms at 50 K. An air-dried film showed similar behavior. The dramatic change of half-time between 300 and 200 K can be ascribed to one or more phase transitions involving water; in a dehydrated film the half-time of the fastest decay component was about 22 +/- 5 ms, independent of the temperature between 300 and 70 K. The original properties of an air-dried film were restored fully, after dehydration, by exposure to either H2O or 2H2O vapor. There was no significant difference, in these measurements, between H2O-restored and 2H2O-restored films.

Bacterial Chromatophores↗
PubMed1978

EPR properties of the reaction center of Rhodopseudomas gelatinosa in situ and in a detergent-solubilized form.

The photochemical reaction centers from a variety of purple photosynthetic bacteria are composed of a trimer of protein subunits. However, the recently isolated reaction center from Rhodopseudomonas gelatinosa appears to have only two subunits. In this paper we examine the EPR characteristics of the primary photochemical reactants in this species, and compare them with those of other species. Despite of the differences in protein composition, no dramatic differences in EPR properties are seen in vivo, although some interesting effects are seen upon solubilization of the reaction center, which may be related to the unusual lability of the isolated preparation. Perhaps the most noteworthy phenomenon seen in Rps. gelatinosa is the apparent ability of electrons on the reduced intermediary electron carrier to tunnel at low temperatures to the oxidized c-type cytochrome, which has not been seen in other species studied to date.

Bacterial Proteins↗
PubMed1978

Properties of photochemical reaction centers purified from Rhodopseudomonas gelatinosa.

Reaction centers were isolated from a carotenoidless mutant of Rhodopseudomonas gelatinosa by hydroxyapatite chromatography of purified chromatophores treated with lauryl dimethyl amine oxide. Absorption spectra and spectra of light-induced absorbance changes are similar to those of reaction centers from Rhodopseudomonas sphaeroides. The ratio of absorbance at 280 nm to that at 799 nm was 1.8 in the purest preparations. The extinction coefficient at the 799 nm absorption maximum was estimated to be 305 +/- 20 mM--1 . CM--1. The molecular weight based on protein and chromophore assays was found to be 1.5 . 10(5); the reaction center protein accounted for 6% of the total membrane protein. These reaction centers contained no cytochrome and showed just two components of apparent molecular weights 33 000 and 25 000 in polyacrylamide gel electrophoresis. The chromatophores contained 42 molecules of antenna bacteriochlorophyll for each reaction center.

Bacterial Chromatophores↗
PubMed1978

Picosecond and steady state, variable intensity and variable temperature emission spectroscopy of bacteriorhodopsin.

The bacteriorhodopsin emission lifetime at 77 degrees K has been obtained for different regions of the emission spectrum with single-pulse excitation. The data under all conditions yield a lifetime of 60 +/- 15 ps. Intensity effects on this lifetime have been ruled out by studying the relative emission amplitude as a function of the excitation pulse energy. We relate our lifetime to previously reported values at other temperatures by studying the relative emission quantum efficiency as a function of temperature. These variable temperature studies have indicated that an excited state with an emission maximum at 670 nm begins to contribute to the spectrum as the temperature is lowered. Within our experimental error the picosecond data seem to suggest that this new emission may arise from a minimum of the same electronic state responsible for the 77 degrees K emission at 720 nm. A correlation is noted between a 1.0-ps formation time observed in absorption by Ippen et al. (Ippen, E.P., C.V. Shank, A. Lewis, and M.A. Marcus. 1978. Subpicosecond spectroscopy of bacteriorhodopsin. Science [wash. D.C.]. 200:1279-1281 and a time extrapolated from relative quantum efficiency measurements and the 77 degrees K fluorescence lifetime that we report.

Bacteriorhodopsins↗
PubMed1977

Ubiquinone reduction and proton uptake by chromatophores of Rhodopseudomonas sphaeroides R-26: periodicity of two in consecutive light flashes.

Chromatophores of Rhodopseudomonas sphaeroides strain R-26 were subjected to a series of brief flashes of light in the presence of diaminodurene as an electron donor. Odd-numbered flashes induced the reduction of ubiquinone to the anionic semiquinone, as indicated by absorbance changes near 450 nm. This reaction was not attended by proton binding. Even-numbered flashes caused disappearance of the semiquinone, presumably by conversion to the fully reduced form. This reaction was attended by proton uptake.

Bacterial Chromatophores↗
PubMed1977

The carotenoid band shift in reaction centers from from Rhodopseudomonas sphaeroides.

A specific carotenoid associated with reaction centers purified from Rhodopseudomonas sphaeroides shows an optical absorbance change in response to photochemical activity, at temperatures down to 35 K. The change corresponds to a bathochromic shift of 1 nm of each absorption band. The same change is induced by either chemical oxidation or photo-oxidation of reaction center bacteriochlorophyll (P-870). Reduction of the electron acceptor of the reaction center, either chemically or photochemically, does not cause a carotenoid absorbance change or modify a change already induced by oxidation of P-870. The change of the carotenoid spectrum can therefore be correlated with the appearance of positive charge in the reaction center. In these studies we observed that at 35 K the absorption band of reaction center bacteriochlorophyll near 600 nm exhibits a shoulder at 605 nm. The resolution into two components is more pronounced in the light-dark difference spectrum. This observation is consistent with our earlier finding, that the "special pair" of bacteriochlorophyll molecules that acts as photochemical electron donor has a dimer-like absorption spectrum in the near infrared.

Bacteriochlorophylls↗