FLASH PHOTOLYSIS-ELECTRON SPIN RESONANCE: A KINETIC STUDY OF ENDOGENOUS LIGHT-INDUCED FREE RADICALS IN REACTION CENTER PREPARATIONS FROM RHODOPSEUDOMONAS SPHEROIDES*

Abstract— Using the technique of flash photolysis-electron spin resonance, we have detected, by means of a kinetic analysis, a rapidly decaying signal in reaction center preparations from the R26 blue-green mutant of Rhodopseudomonas spheroides. This signal, which we designate Signal B3, is essentially the same as that seen previously in iron-free preparations. Signal B3 decays at 20°C with a 1/ e time of ˜ 3 ms and exhibits an activation energy of 5 ± 1 kcal mol^-1 over the temperature range 0–30°C. Extraction with isooctane completely eliminates Signal B3, whereas readdition of exogenous ubiquinone-30 completely restores the signal. o -Phenanthroline has no effect on Signal B3. We discuss these results in terms of a model in which the primary acceptor is an iron-ubiquinone complex with excess ubiquinone serving as a secondary electron acceptor pool.     

CONTROL OF SYNTHESIS OF REACTION CENTER BACTERIOCHLOROPHYLL IN PHOTOSYNTHETIC BACTERIA

Abstract— The ratio of total bacteriochlorophyll (BChl) to reaction center BChl has been determined in the wild-type and carotenoid-less mutants of Rhodopseudomonas spheroides and Rhodospirillum rubrum with various specific BChl contents. In wild-type R. spheroides the ratio increases as the specific BChl content increases; in the other strains the ratio is almost invariant.
The ability of the wild-type of R. spheroides to alter the size of the photosynthetic unit is apparently correlated with its multi-component BChl spectrum. The amount of reaction center BChl relative to the amount of the longest wavelength component of the spectrum is constant. The variation in the size of the photosynthetic unit is due to variation in the amount of this component relative to total BChl.
The size of the photosynthetic unit does not change as the specific BChl content decreases during growth under aerobic conditions of a culture grown previously under photosynthetic conditions.     

LIGHT-INDUCED ABSORBANCY CHANGES IN EIMHJELLEN''S RHODOPSEUDOMONAS*

Abstract— Light induced absorbancy changes in Rhodopseudomonas sp. NHTC 133 are analogous to those observed in other photosynthetic bacteria, but all of the features in the light-induced difference spectrum (except those reflecting cytochrome oxidation) are shifted to greater wavelengths. This general shift is consistent with the fact that the bacteriochlorophyll of this organism (BChl b ) differs from the usual BChl in that its absorption bands are shifted to greater wavelengths.
The singlet excited state of P985 (the counterpart of the P870 of Rhodopseudomonas spheroides ) is not an energy sink relative to the major component of BChl b in vivo . The absorption maximum of P985 is at 985 mp, whereas that of CBhl b in vivo is at 1012 mμ.     

EFFECTS OF HIGH PRESSURE ON PHOTOCHEMICAL REACTION CENTERS FROM RHODOPSEUDOMONAS SPHEROIDES*

Abstract— Photochemical reaction centers from Rhodopseudomonas spheroides were subjected to pressures ranging from 1 to 6000 atm. Optical absorption, fluorescence and photochemical activities were studied under these conditions. Absorption spectra showed bathychromic shifts of the long-wave bands attributed to bacteriopheophytin and bacteriochlorophyll (the latter as P800 and P870). The quantum efficiency of photochemical oxidation of P870 was diminished at high pressure. The quantum yield of P870 fluorescence showed a parallel decline, as if high pressure introduced a quenching process that competed with both photochemistry and fluorescence. The original efficiencies were largely restored when the pressure was returned to 1 atm. The efficiency of oxidation of mammalian cytochrome c, coupled to the photochemical oxidation of P870 in reaction centers, was lowered by high pressure. This effect was more pronounced than the effect on P870 oxidation, and was irreversible. The kinetics of recovery of P870 following its photochemical oxidation showed effects of high pressure. The main effect was the appearance, at high pressure, of slow recovery suggesting the trapping of electrons. This effect was partly irreversible.     

FLASH PHOTOLYSIS-ELECTRON SPIN RESONANCE STUDIES OF THE INTERACTION OF PHENAZINE METHOSULFATE WITH REACTION-CENTER PREPARATIONS FROM THE R-26 MUTANT OF RHODOPSEUDOMONAS SPHEROIDES*

Abstract— Using the technique of flash photolysis-electron spin resonance, we have shown, by means of a kinetic analysis, that phenazine methosulfate (PMS) interacts with reaction-center preparations from the blue-green mutant R26 of Rhodopseudomonas spheroides. At intermediate concentrations of PMS, biphasic decay kinetics of the P870⁺ ESR signal are observed demonstrating that the PMS radical interacts with reaction centers by a specific binding mechanism. With PMS bound to reaction centers, the P870⁺ ESR signal decays in ˜ 1 ms; whereas, in unbound reaction centers the decay is ˜ 120 ms. A model is proposed involving the interaction of PMS on the donor side of P870.     

SPECTRA AND EXTINCTION COEFFICIENTS OF NEAR-INFRARED ABSORPTION BANDS IN MEMBRANES OF Rhodobacter sphaeroides MUTANTS LACKING LIGHT-HARVESTING AND REACTION CENTER COMPLEXES

Abstract— Membranes from Rhodobacter (formerly Rhodopseudomonas) sphaeroides mutant strains that lack one or more of the bacteriochlorophyll a (BChl)-protein complexes were used to obtain spectra and molar extinction coefficients of the near-IR absorption bands. The strains examined were NF57 which lacks the B875 light-harvesting and reaction center complexes, and M21 which lacks the B800–850light-harvesting complex. The extinction coefficients obtained for the B800, B850 and B875 bands were 226 ± 10, 170 ± 5, and 118 ± 5 m M ^-1cm^-1, respectively, in reasonable agreement with values reported for detergent-isolated complexes (Clayton, R. K. and B. J. Clayton, Proc. Natl. Acad. Sci. USA 78 ,5583–5587, 1981). The results also demonstrated that detergent solubilization altered the spectra, causing a band broadening on the blue side of the B875 and B800 peaks. The data obtained from the analysis of the mutant strains were used for deconvolution of the BChl species in membranes of the wild-type. A short BASIC computer program for performing this deconvolution is included.     

LINEAR AND CIRCULAR DICHROISM AND FLUORESCENCE POLARIZATION OF THE B875 LIGHT-HARVESTING BACTERIOCHLOROPHYLL-PROTEIN COMPLEX FROM RHODOPSEUDOMONAS SPHAEROIDES

Abstract— The orientation of the chromophores in the B875 light-harvesting bacteriochlorophyll complex isolated from Rhodopseudomonas sphaeroides by lithium dodecyl sulfate/polyacrylamide gel electrophoresis was examined by linear and circular dichroism and fluorescence polarization procedures. The circular dichroism in the near-IR was weaker than that of the B800–850 light-harvesting complex and had a distinctly different shape. This suggested a different geometry for the two bacteriochlorophylls of B875 and less interactive association between their transition moments. The magnitude of the circular dichroism in the carotenoid region of B875 was similar to that of B800–850 but gave more negative values between approx. 430–485 nm; this may reflect a difference in the asymmetric binding of carotenoids to the B875 protein. The fluorescence polarization increased sharply across the near-IR region of B875 and achieved very high values at long wavelengths. This confirmed that more than one transition contributed to this absorption band. The linear dichroism of B875 did not show a significant change in this near-IR band like that observed for the longest wavelength band of B800–850. Thus, the transition moments for each bacteriochlorophyll within B875 appear to be tilted to approximately the same extent with respect to the protein axis. These results distinguish B875 from all other light-harvesting complexes and suggest that the antennae of Rhodospirillaceae which contain a single near-IR absorption band cannot be classified into a single group.     

PHOTOCHEMICAL QUANTUM EFFICIENCY AND ABSORPTION SPECTRA OF REACTION CENTERS FROM RHODOPSEUDOMONAS SPHAEROIDES AT LOW TEMPERATURE

Abstract— The quantum efficiency of bacteriochlorophyll photo-oxidation in reaction centers from Rhodopseudomonas sphaeroides is independent of temperature, within experimental accuracy of ± 15%, from 300 K to 5 K. Absorption spectra at low temperature show maxima at 532 and 544 nm corresponding to the two molecules of bacteriopheophytin in the reaction center. Comparison with the spectrum of the transient light-induced state P ^F indicates that of these two molecules, only the one absorbing at 544 nm participates in forming P ^F.     

CAROTENOID PIGMENTS AND THE IN VIVO SPECTRUM OF BACTERIOCHLOROPHYLL

Abstract— The isolation of a mutant, strain PM-9, of Rhodopseudonionus spheroides with an abnormal complement of carotenoid pigments is described.
PM-9 accumulates phytoene, phytofluene, ζ-carotene and neurosporene. Semi-aerobic cultures form more ζ-carotene and neurosporene relative to total carotenoids than do photo-synthetic cultures.
PM-9 is killed on exposure to light and oxygen.
By making use of the effect of oxygen on the nature of the carotenoids in PM-9, we have shown that these pigments do not directly influence the in vivo spectrum of bacteriochlorophyll.
Diphenylamine inhibits the synthesis of coloured carotenoids in Rhodopseudonionos gelatinosa but does not change the bacteriochlorophyll spectrum.     

THE EFFECT OF PHOSPHORYLATION UNCOUPLERS AND ELECTRON TRANSPORT INHIBITORS UPON SPECTRAL SHIFTS AND DELAYED LIGHT EMISSION OF PHOTOSYNTHETIC BACTERIA

Abstract— Delayed light emission (measured 4 msec after excitation) and the light-induced red shifts of the bacteriochlorophyll and carotenoid absorption bands of chromatophores of Rhodopseudomonas spheroides were inhibited by a variety of reagents. These included anti-mycin A, NQNO, CCCP, desaspidin, quinacrine, chlorpromazine, 2,4-dinitrophenol, gramicidin D, Triton X-100 and valinomycin in the presence of potassium, cesium or ammonium ions. Delayed light emission was enhanced by orthophenanthroline, ethanol, succinate and glutathione.
Delayed light emission from chromatophores of Rhodospirillum rubrum was attenuated during photophosphorylation but restored approximately to its initial value in the presence of oligomycin. Since the delayed light and band shifts are inhibited under conditions which tend to deplete or block the formation of high energy phosphorylation intermediate, it is suggested that the presence of a high energy intermediate is a prerequisite for the appearance of each of the three phenomena.     

RELATIONS BETWEEN PHOTOCHEMISTRY AND FLUORESCENCE IN CELLS AND EXTRACTS OF PHOTOSYNTHETIC BACTERIA*

Abstract— Light-induced changes in the yield of bacteriochlorophyll fluorescence have been measured in cells and chromatophores of photosynthetic bacteria, and coordinated with light-induced absorbancy changes. Comparisons were drawn during transitions between dark and light steady states and also between steady states established at different light intensities. Aerobic cell suspensions of Rhodospirillum rubrum, Rhodopseudomonas spheroides, Chromatium and Rhodopseudomonas sp. NHTC 133 showed a strict correspondence between changes in the fluorescence yield and the bleaching of P870 (P985 in Rps. sp. NHTC 133), as reported by Vredenberg and Duysens for R. rubrum cells. The relationship shows that singlet excitation energy in bacteriochlorophyll is quenched by P870 at a rate proportional to the concentration of unbleached P870. This implies that the photosynthetic units are not independent with respect to energy transfer. In anaerobic cell suspensions the change in fluorescence did not follow the bleaching of P870 in the manner described by Vredenberg and Duysens. Here a change in fluorescence may have resulted from the reduction of a primary photochemical electron acceptor as well as from the oxidation (bleaching) of P870. In chromatophore preparations there were further deviations from the Vredenberg and Duysens relationship which could be attributed to changes in the rate constants for quenching of singlet excitation energy. Finally there was a light-induced increase in the fluorescence yield which was related to a band shift of bacteriochlorophyll and not to the bleaching of P870. Aerobic cell suspensions presented a limiting case in which these complications were absent. No change in the fluorescence was associated uniquely with the oxidation of cytochrome or band shifts of carotenoid pigments. These results, when coordinated with earlier findings about the fluorescence of bacteriochlorophyll and P870, indicate that the singlet excitation quantum is the only energy carrier linking the absorption of light with the initiation of photochemistry in bacterial photosynthesis.     

Structure, function and organization of antenna polypeptides and antenna complexes from the three families of Rhodospirillaneae.

Comparative primary structural analysis of polypeptides from antenna complexes from species of the three families of Rhodospirillaneae indicates the structural principles responsible for the formation of spectrally distinct light-harvesting complexes. In many of the characterized antenna systems the basic structural minimal unit is an alpha/beta polypeptide pair. Specific clusters of amino acid residues, in particular aromatic residues in the C-terminal domain, identify the antenna polypeptides to specific types of antenna systems, such as B880 (strong circular dichroism (CD)), B870 (weak CD), B800-850 (high), B800-850 (low) or B800-820. The core complex B880 (B1020) of species from Ectothiorhodospiraceae and Chromatiaceae apparently consists of four (alpha 1 alpha 2 beta 1 beta 2) or three (2 alpha beta 1 beta 2) chemically dissimilar antenna polypeptides respectively. There is good evidence that the so-called variable antenna complexes, such as the B800-850 (high), B800-850 (low) or B800-820 of Rp. acidophila, Rp. palustris and Cr. vinosum, are comprised of multiple forms of peripheral light-harvesting polypeptides. Structural similarities between prokaryotic and eukaryotic antenna polypeptides are discussed in terms of similar pigment organization. The structural basis for the strict organization of pigment molecules (bacteriochlorophyll (BChl) cluster) in the antenna system of purple bacteria is the hierarchical organization of the alpha- and beta-antenna polypeptides within and between the antenna complexes. On the basis of the three-domain structure of the antenna polypeptides with the central hydrophobic domain, forming a transmembrane alpha helix, possible arrangements of the antenna polypeptides in the three-dimensional structure of core and peripheral antenna complexes are discussed. Important structural and functional features of these polypeptides and therefore of the BChl cluster are the alpha/beta heterodimers, the alpha 2 beta 2 basic units and cyclic arrangements of these basic units. Equally important for the formation of the antenna complexes or the entire antenna are polypeptide-polypeptide, pigment-pigment and pigment-polypeptide interactions.     

ISOLATION AND CHARACTERIZATION OF A STRUCTURAL SUBUNIT FROM THE CORE LIGHT-HARVESTING COMPLEX OF Rhodobacter sphaeroides 2.4.1 AND puc705-BA

A method for isolating a structural subunit, B825, from the B875 core light-harvesting complex (LHC) of Rhodobacter sphaeroides 2.4.1 (wild-type) and a B800-B850(-) mutant, puc705-BA, is presented. This method, based on one developed to prepare a similar subunit, B820, from the core LHC of Rhodospirillum rubrum [Miller et al., Biochemistry 26, 5055-5062 (1987)], requires the dissociation of treated chromatophores with the detergent, octyl-glucoside. A subsequent gel filtration step separates B800-850 (if present), reaction centers, and free bacteriochlorophyll from the subunit complex. B825 was quantitatively reassociated into an 873 nm absorbing form which resembled the in vivo complex as judged by its absorption properties. The polypeptides in B825 and B800-850 were isolated by HPLC and identified by N-terminal amino acid sequencing. Two polypeptides, alpha and beta, were found in each complex in a 1:1 ratio. The spectral and biochemical properties of the subunits isolated from Rhodospirillum rubrum, Rhodobacter sphaeroides, and Rhodobacter capsulatus are compared.     

SPECTRAL MODIFICATIONS OF BACTERIAL ANTENNA COMPLEXES BY LIMITED PROTEOLYSIS

The spectral effects of a number of endo- and exoproteases to the detergent-solubilized antenna complexes B802–858(sphaeroides like) of strain Rhodopseudomonas acidophila 10050 and B802–824of strain 7050 were analyzed. Upon protease digestion the time course of spectral modification between 250 and 900 nm was recorded. In addition, circular dichroism (CD) signals were measured between 450 and 950 nm. The endoprotease elastase as well as the exoprotease carboxypeptidase A (CP A) altered the B802–858antenna complex, by changing the 858 nm band hypochromically (30-50%) with an additional small hypsochromic shift (3–6nm). A combined carboxypeptidase digestion, e.g. CP A and CP B, yielded a further modification to a complex with absorption bands at 800 nm and ?840 nm (ε? 50% of the 800 nm band). By exposing the isolated B803–824antenna complex to the different proteases no significant spectral change was observed. The B802–858antenna complex, modified by CP A, exhibits a60–70% decrease of the biphasic CD signal in the near infrared. The limited proteolysis experiments provide conclusive experimental evidence that the C-terminal domains of the antenna polypeptides α and β contribute to the formation of (dimeric) bacteriochlorophyll (BChl) a molecules absorbing at around 850 nm. This agrees with the hypothesis, based on comparative amino acid sequence analysis, that in purple bacterial antenna complexes the structural requirements (specific amino acids in the vicinity of the BChl molecules, e.g. aromatic amino acids) for batho- and hyperchromicity of BChl molecules apparently reside to a considerable extent in the C-terminal portions of their antenna apoproteins.     

REVERSIBLE OXIDATION OF ANTENNA BACTERIOCHLOROPHYLL IN TWO PHOTOREACTION CENTERLESS MUTANTS OF RHODOSPIRILLVM RUBRUM

Abstract— Reversible oxidation of bacteriochlorophyll in two independent mutants of Rhodospirillum rubrum lacking photoreaction center has been studied to obtain further information about the antenna system of that bacterium. Oxidation of antenna bacteriochlorophyll with ferricyanide is accompanied by three distinct absorbance changes in the near infrared: the appearance of a new band at 1230 nm (B1230) and the bleaching of two bands, a major one at 882 nm (B882) and a minor one at 888 nm (B888). The three absorbance changes take place at potentials higher than those required for complete oxidation of photoreaction-center bacteriochlorophyll in wild-type strains. On the other hand, the B882 bleaching and the B1230 appearance occur under similar redox conditions. The above data suggest, first, that in Rds. rubrum , contrary to the current generalized idea, antenna bacteriochlorophyll exhibits at least two absorption bands, B888 and B882; second, that B882 and B1230 correspond to the same bacteriochlorophyll component. In addition, the appearance of B1230 may indicate that antenna bacteriochlorophyll can be partially arranged in an oligomeric fashion, for bands in that spectral region are not exhibited by oxidized monomeric bacteriochlorophyll.     

FLUORESCENCE YIELD PROPERTIES OF A FRACTION ENRICHED IN NEWLY SYNTHESIZED BACTERIOCHLOROPHYLL U-PROTEIN COMPLEXES FROM RHODOPSEUDOMONAS SPHAEROIDES

Abstract— A membrane fraction enriched in newly synthesized bacteriochlorophyll a-protein complexes was isolated from Rhodopseudomoms sphaeroides by rate-zone sedimentation. An examination of the fluorescence yield properties showed that the ratio of the maximal fluorescence emission near 910 nm (with all photochemical traps closed) to that of the initial fluorescence rise (with all traps open) was 2.2 compared to 2.9 in chromatophores. The spectrum for the variable portion of the fluorescence emission (the slow rise between the initial and maximal levels) was essentially the same in both fractions, but that observed for the initial rise in the newly synthesized material showed a greater fluorescence yield with a broad peak near 865 nm. This extra emission is thought to arise from the light-harvesting bacteriochlorophyll complex with an absorption maximum at 850 nm and suggests that this component is only partially connected to photosynthetic units. In contrast, the little extra emission observed at the longer wavelengths in this fraction indicated that energy is transferred more efficiently between the 875 nm antenna complex and photochemical reaction centers. The kinetics of the fluorescence rise suggest that photosynthetic units exist at separate sites in newly synthesized membrane regions.     

ISOLATION and SPECTROSCOPIC CHARACTERIZATION OF THE B875 ANTENNA COMPLEX OF A MUTANT OF Rhodopseudomonas sphaeroides

Abstract— We describe a procedure of purification of the B875 antenna complex isolated from the 3P17 mutant strain of Rhodopseudomonas sphaeroides, enriched in B875. The integrity of this isolated complex, as well as a very low content of residual B800-850 antenna, was suggested from low temperature absorption and resonance Raman spectra. Time resolved experiments were also carried out. The important result is the identity of the fluorescence lifetime of the B875 isolated complex (0.64 ± 0.03 ns) with that of the B875 antenna in vivo (0.63 ns), in the membrane of the C71 reaction center-less mutant of Rhodopseudomonas sphaeroides, measured in our previous study.
Our data suggest that the interactions between the bacteriochlorophylls of the complex, as well as the constraints imposed by their protein environment are not much changed from the in vivo state.     

Molecular assembly of Zn porphyrin complexes onto a gold electrode using synthetic light-harvesting model polypeptides

Molecular assembly of Zn porphyrin pigments, Zn substituted bacteriochlorophyll a ([Zn]-BChl a) and Zn mesoporphyrin monomer (ZnMPMME) on a gold electrode using synthetic 1α-helix hydrophobic polypeptides which have similar amino acid sequences to the hydrophobic core in the native photosynthetic light-harvesting (LH) 1-β polypeptide from Rhodobacter sphaeroides, has been achieved: this process is dependent on the structures of pigments and polypeptides. Interestingly, an enhanced photoelectric current was observed when ZnMPMME with the LH1 model polypeptide in an α-helical configuration was assembled onto the electrode.     

ENERGY TRANSFER AND PHOTOOXIDATION KINETICS IN REACTION CENTERS ON THE PICOSECOND TIME SCALE

Abstract— Picosecond 530 nm actinic and 1242 nm probe light pulses have been used to measure the kinetics of energy transfer and photooxidation in Rhodopseudomonas sphaeroides R-26 reaction centers. The energy transfer rate between bacteriopheophytin and the bacteriochlorophyll dimer is 1.0 ± 0.3 ± 10¹¹s^-land photooxidation of the dimer occurs within 5 ps after the dimer reaches the first excited singlet state. Using these parameters in a simple model we are able to explain the odd result that the number of reaction centers oxidized by a saturating 530 nm actinic picopulse is only 60% of the number oxidized by a saturating CW light source.     

VARIATION IN ELECTRON PARAMAGNETIC RESONANCE SIGNALS OF PHOTOSYNTHETIC SYSTEMS WTTH THE REDOX LEVEL OF THEIR ENVIRONMENT

Abstract— The redox dependence of the light-induced electron paramagnetic resonance signal at g=2 in R. rubrum, R. spheroides and Chromatium chromatophore particles and quantasonie particles from spinach chloroplasts has been determined qualitatively over the range —0.3 to +0.6 V and quantatively over the range +0.3 to ±0.6 V. A light-induced EPR signal has been titrated and demonstrated to have a midpoint potential of +0.44 v at pH 7 and 20°C. Concentration, ionic strength and pH dependence for this transition in R. rubrum chromatophores is reported. In addition to the dark signal which replaces the light signal, in chromatophore material another dark signal, occurring in the seine location as the light signal, has been demonstrated to occur at high potential.
Selective chemical oxidation with K₂lrCl₆ of chromatophore particles from the three bacteria resulted in the removal of some 95 per cent of the absorbance in the near infrared and left the photoactive pigments.
Two light-induced EPR signals were found in quantasome particles by their dependence upon the redox level. Of particular interest is a signal observed at quite high potential (e.g. + 0.60 V). It was demonstrated that oxygen evolution by these quantasonie particles in the presence of K³Fe(cN)₀ occurred at the same rate at +0.55 V as at +0.40 V.