Gabriel Gingras

Structure and function of carotenoids in the photoreaction center from Rhodospirillum rubrum

Abstract The bacteriochlorophyll ( P -800 and P -870) of the carotenoidless photoreaction center isolated from Rhodospirillum rubrum (strain G9) is bleached irreversibly when the preparations are exposed to intense near infrared light in the presence of oxygen. This effect is much smaller in preparations, extracted from the wild type, which contain, as shown earlier, 1 mol of spirilloxanthin per mol of P -870. This photodynamic effec is shown to be due to singlet O 2 . The oxidation of adrenaline in the presence of superoxide dismutase and the oxidation of 1,3-diphenylisobenzofuran are used as reporter reactions. Singlet oxygen is presumably generated by the triplet-triplet energy transfer 3 bacteriochlorophyll → O 2 ( 3 Σ ). Four purified bacterial carotenoids, spirilloxanthin, sphaeroidene, sphaeroidenone and chloroxanthin were attached onto the carotenoidless photoreaction center from strain G9 in nearly 1 : 1 mol ratios with respect to P -870. Once fixed, these carotenoids confer protection against the photodynamic bleaching of bacteriochlorophyll. The relative photoprotection efficiency was 1.0 for spirilloxanthin and sphaeroidene, 0.4 for chloroxanthin and 0.2 for sphaeroidenone. The fixed carotenoids display optical activity and their molar ellipticity appears to be correlated with their relative photoprotection efficiency. The efficiency of energy transfer to P -870 is 0.90 for sphaeroidene, 0.35 for sphaeroidenone, 0.30 for chloroxanthin and 0.20 for spirilloxanthin. The energy transfer efficiency from the carotenoids to bacteriochlorophyll is suggested to be governed by the rate of the internal conversion processes of the excited singlet state of the carotenoids. A study of the difference absorption and CD spectra of the reconstituted minus carotenoidless preparations leads to the interpretation that the fixed carotenoids are in a central monocis conformation.

Isolation and partial characterization of a P870 reaction center complex from wild type Rhodospirillum rubrum

P870 was isolated from wild type Rhodospirillum rubrum by solubilization with dodecyldimethylamine oxide followed by ammonium sulfate precipitation. The particle contains some carotenoids, 0.3 mole of ubiquinone per mole of P870 and no cytochrome. The apparent particle weight is 140 000. Electrophoresis in dissociating media containing 0.1% sodium dodecyl sulfate shows the existence of at least two and more likely three protein bands. The complex is calculated to have a minimal molecular weight of 70 600 ± 3200 and is supposed to be formed of three protomers.

SPECTRAL EVIDENCE FOR PHOTO‐INDUCED ISOMERIZATION OF CAROTENOIDS IN BACTERIAL PHOTOREACTION CENTER

Abstract The aim of this work was to determine whether spirilloxanthin and sphaeroidene bound to the same site of the photoreaction center isolated from Rhodospirillum rubrum can display cis–trans isomerization under conditions that lead to formation of triplet state PR of the primary electron donor. To this end, we monitored changes in the absorption spectrum of these bound carotenoids as induced by red light at low redox potentials. This experiment was performed both with the intact photoreaction center isolated from strain Sl and with the photoreaction center isolated from carotenoidless strain G9 reconstituted with either spirilloxanthin or sphaeroidene. In both preparations, spirilloxanthin exhibited light‐induced absorption changes that can be interpreted as a cis–trans isomerization. Under our experimental conditions, the absorption changes attained their full extent in about 1 min and were not or were only partially reversed when the light was switched off. Under our experimental conditions, the extent of these changes indicate that about 15% of the bound spirilloxanthin undergoes isomerization. Sphaeroidene artificially attached to the G9 photoreaction center also undergoes light‐induced absorbance changes, but these cannot easily be interpreted as a cis–trans isomerization.

Chemical and physical properties of a carotenoprotein from Rhodospirillum rubrum

Abstract A carotenoprotein, obtained from chromatophores of Rhodospirillum rubrum by solubilization with sodium dodecylsulfate, was purified by (NH4)2SO4 fractionation followed by gel filtration. Immunodiffusion and polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate indicate the preparation to be highly purified. The complex contains about 80% protein plus carbohydrate, spirilloxanthin and possibly phospholipid. The results indicate that spirilloxanthin is bound non-covalently in a 1:1 fashion with a single polypeptide chain. The apparent molecular weight of this complex was found to be 11 000. It is composed of 52% hydrophobic amino acid residues and calculated to represent at least 10% of the membrane proteins. On the basis of absorption and CD spectroscopy, it is proposed that the interaction of the carotenoid with the protein brings about a twisting of the former around its central double bond. The angle of twisting would be sufficient to break the conjugated double bond system into two halves.

Photodichroic studies of the photoreaction center from Rhodospirillum Rubrum. I. Attribution of P870 to two non parallel dipoles

A randomly oriented sample of photoreaction center prepared from Rhodospirillum rubrum was excited at 77 degrees K by an actinic linearly polarized light of 870 nm. Under such conditions, only those chromophores with components of their absorption dipoles oriented parallel to the polarization of the actinic light are bleached. The change in absorbance at 900 nm of this photoselected sample was observed while varying the angle of polarization of a weak measuring light. The polarization of the absorbance change was thus evaluated as 0.25. This value is interpreted to mean that P870 is attributable to two absorption dipoles forming an angle included between 35.75 degrees and 90 degrees. Comparison with the p value of 0.5 obtained on a similar preparation by polarization of fluorescence (Ebrey, T. G. and Clayton, R.K. (1969) Photochem. Photobiol. 10, 109-117) leads to the conclusion that either these two dipoles emit fluorescence without being coupled by singlet-singlet energy transfer or that only one of them is a fluorescence emitter in the absence of reversible singlet-singlet energy transfer.

A carotenoprotein from chromatophores of Rhodospirillum rubrum

Abstract A carotenoprotein has been obtained by SDS-solubilization of Rhodospirillum rubrum chromatophores. It was then purified by (NH4)2SO4 precipitation and Sephadex G-200 filtration. SDS-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of about 12,000. The absorption spectrum of the complex is entirely different from the usual three peaked carotenoid spectrum, it has only a major peak at 370 nm. However, after acetone extraction the spectrum of spirilloxanthin reappears. The fact that the carotenoid associates with a specific protein provides strong evidence that the complex originates from the chromatophores and is not a preparative artefact.

Relative phototrapping rates of the two bacteriopheophytins in the photoreaction center of Ectothiorhodospira sp.

The photoreaction center of Ectothiorhodospira sp. includes a bound c-type cytochrome containing four hemes. When reduced, this cytochrome competes with the reduced primary electron acceptor for charge recombination with the oxidized primary donor. This allows the phototrapping of reduced intermediary species. While only the A arm bacteriopheophytin anion was phototrapped at temperatures above 250 K, both bacteriopheophytin anions were phototrapped at 219 K. The trapping and decay rate constants of the two bacteriopheophytin anions were obtained from the kinetics were obtained from the kinetics of their light-induced absorbance change and EPR signal. The bacteriopheophytin situated in the A arm was phototrapped 274-times faster than the one in the B arm. Both phototrapped anions decay by charge recombination with the oxidized cytochrome.

An asymmetric dimer exciton model. Application to the primary electron donor of bacterial photoreaction center

Abstract An asymmetric dimer excition theory is developed that takes into account both environmental and vibronic effects on the electronic transition energies. Explicit equations are presented for the transition energies, the dipole moments, the angle between the dipole moments and the dipole and rotational strengths for the electronic transitions in this asymmetric dimer. This model is proposed to describe the structure of the special pair of bacteriochlorophyll a molecules believed to constitute the primary electron donor of bacterial photoreaction center. The model is found to be consistent with most of the spectroscopic properties of the photoreaction center. We used the equations derived from the asymmetric model along with absorption and circular dichroism spectroscopy data to predict a geometrical structure for the primary electron donor.

Different temperature dependencies of the charge recombination reaction in photoreaction centers isolated from different bacterial species

Abstract We compared the temperature dependency of the rate of the charge recombination reaction in photoreaction centers isolated from Ectothiorhodospira sp. and from Rhodospirillum rubrum G9. We also examined the temperature dependency of the bandwidth and peak wavelength of their far-red absorption band. In both preparations, the peak wavelength and the bandwidth vary monotonically with temperature between 80 and 300 K. However, the rate of the charge recombination reaction has a quite different temperature dependency. In the preparation from R. rubrum, the reaction is accelerated 5-fold in a typical sigmoidal fashion as the temperature is lowered from 300 to 80 K. In the preparation from Ectothiorhodospira sp., the reaction is accelerated monotonically only about 1.5-fold in the same temperature range. At temperatures below 100 K, the rates are similar in the two preparations. We interpret the temperature dependency of the charge recombination reaction in terms of an activationless electron-transfer model formulated by Jortner (Jortner, J. (1980) Biochim. Biophys. Acta 394, 193–230). The minimal model provides a good fit for the temperature dependency of charge recombination in the preparation from Ectothiorhodospira sp. However, to fit the temperature dependency of the R. rubrum preparation with the same model, we must further postulate that the electronic coupling factor varies with temperature in this preparation. We find that, in both preparations, the temperature dependency of the far-red absorption bandwidth is consistent with the assumption that similar vibrational modes are involved in electron transfer and in electronic excitation.

The photogeneration of superoxide by isolated photoreaction center from Rhodospirillum rubrum.

Abstract The photochemical activity of isolated photoreaction center is accompanied by the formation of superoxide radical anions. This is shown by the photoinduction of the aerobic chain oxidation of sulfite or of adrenalin and by the reduction of ferricytochrome c. These phenomena can be inhibited by superoxide dismutase. Since 0-phenanthroline does not affect the formation of superoxide, the photoreduction of oxygen is supposed to take place on or before the primary electron acceptor.