Roderick K. Clayton

Pigment content and molar extinction coefficients of photochemical reaction centers from Rhodopseudomonas spheroides

Reaction center particles isolated from carotenoidless mutant Rhodopseudomonas spheroides were studied with the aim of determining the pigment composition and the molar extinction coefficients. Two independent sets of measurements using a variety of methods show that a sample with A800 nm = 1.00 contains 20.8 ± 0.8 μM tetrapyrrole and that the ratio of bacteriochlorophyll to bacteriopheophytin is 2:1. Measurements were made of the absorption changes attending the oxidation of cytochrome c coupled to reduction of the photooxidized primary electron donor in reaction centers, using laser flash excitation. The ratio of the absorption change at 865 nm (due to the bleaching of P870) to that at 550 nm (oxidation of cytochrome) was found to be 5.77. These results, combined with other data, yield a pigment composition of 4 bacteriochlorophyll and 2 bacteriopheophytin molecules in a reaction center. Based on this choice, extinction coefficients are determined for the 802- and 865-nm bands: e802 nm = 288 (± 14) mM−1 · cm−1 and e865 nm = 128 (± 6) mM−1 · cm−1. For reversible bleaching of the 865-nm band, Δered - ox865nm = 112 (± 6) mM−1 · cm−1 (referred to the molarity of reaction centers). Earlier reported values of photochemical quantum efficiency are recomputed, and the revised values are shown to be compatible with those obtained from measurements of fluorescence transients.

The absolute quantum efficiency of bacteriochlorophyll photooxidation in reaction centres of Rhodopseudomonas spheroides.

The widely assumed correspondence between fluorescence and photochemistry in photosynthetic systems has recently been challenged by observations on the triplet state of bacteriochlorophyll in reaction centres of Rhodopseudomonas spheroides. In order to check this assumption we have conducted a precise determination of the quantum efficiency of bacteriochlorophyll photooxidation in reaction centres at room temperature. We find a quantum efficiency of 1.02 +/- 0.04 in contrast to a value of about 0.7 predicted from the variations in fluorescence yield.

Excited states of photosynthetic reaction centers at low redox potentials

In preparations of photochemical reaction centers from Rhodopseudomonas spheroides R-26, lowering the recox potential so as to reduce the primary electron acceptor prevents the photochemical transfer of an electron from bacteriochlorophyll to the acceptor. Measuring absorbance changes under these conditions, we found that a 20-ns actinic flash converts the reaction center to a new state, P-F, which then decays with a half-time that is between 1 and 10 ns at 295 degrees K. At 25 degrees K, the decay half-time is approx. 20 ns. The quantum yield of state P-F appears to be near 1.0, both at 295 and at 15 degrees K. State P-F could be an intermediate in the photochemical electron-transfer reaction which occurs when the acceptor is in the oxidized form. Following the decay of state P-F, we detected another state, P-R, with a decay half-time of 6 mus at 295 degrees K and 120 mus at 15 degrees K. The quantum yield of state P-R is approx. 0.1 at 295 degrees K, but rises to a value nearer 1.0 at 15 degrees K. The kinetics and quantum yields are consistent with the view that state P-R forms from P-F. State P-R seems likely to be a side-product, rather than an intermediate in the electron-transfer process. The decay kinetics indicate that state P-F cannot be identical with the lowest excited singlet state of the reaction center. One of the two states, P-F or P-R, probably is the lowest excited triplet state of the reaction center, but it remains unclear which one.

[69] Photochemical reaction centers from Rhodopseudomonas spheroides

Publisher Summary This chapter discusses the photochemical reaction centers from Rhodopseudomonas spheroids . The hallmark of the known bacterial photosynthetic reaction centers is the presence of a bacteriochlorophyll (BChl) molecule that is specialized to act as a primary photochemical electron donor. This BChl, called “P870,” “P890,” and so on, after the wavelength of its long-wave absorption maximum, is oxidized by light, while an unspecified electron acceptor becomes reduced. Oxidation of P870 is signaled by loss of the long-wave absorption band (bleaching). In living cells, the oxidized P870 is reduced by one or more c -type cytochromes. In purified reaction centers, the source of electrons for the re-reduction of oxidized P870 depends on the environment. In any case, the defining assay for reaction centers is the reversible light-induced bleaching of P870. In the reaction centers made from Rhodopseudomonas spheroides or Rhodospirillum rubrum , the bleaching of P870 is accompanied by blue shift of a band near 800 nm because of another specialized BChl, P800. There appear to be two or three P800 for each molecule of P870. The absorption increase at 780 nm caused by this blue shift is often easier to measure than the bleaching at 870 nm. Rhodopseudomonas viridis contains the longer-wave pigment BChl b . Reaction centers made from this organism contain P830 and P960, analogous to the P800 and P870 of Rhodopseudomonas spheroides . The blue shift of P830 can be measured as an increase in optical density at 810 nm.

Kinetics of electron transfer between the primary and the secondary electron acceptor in reaction centers from Rhodopseudomonas sphaeroides

Photoreduction of the two ubiquinone molecules, UQ1 and UQ2, bound to purified reaction center from Rhodopseudomonas sphaeroides induces different absorption band shifts of bacteriochlorophyll and bacteriopheophytin molecules depending on which ubiquinone is photoreduced. This allows us to study electron transfer between UQ1 and UQ2 directly by absorption spectrometry. The results support a model in which electrons are transferred one by one from UQ1 to UQ2 with a half-time 200 micro seconds, and two by two from fully reduced UQ2 to the secondary acceptor pool.

Relations between pigments and proteins in the photosynthetic membranes of Rhodopseudomonas spheroides

We have isolated from Rhodopseudomonas spheroides a pigment-protein complex of apparent weight 9 kdaltons that bears more than 60% of the light harvesting bacteriochlorophyll. The isolation procedure involved exposure to 1% lauryl dimethyl amine oxide (LDAO). The purified 9-kdalton fraction showed the light harvesting bacteriochlorophyll components B800 and B850, plus carotenoids. The ratio of bacteriochlorophyll to protein was 17%. This protein is probably the same as the “band 15” protein of Fraker and Kaplan. It may exist in vivo as characteristic aggregates of higher molecular weight. LDAO added to Rps. spheroides chromatophores converted the bacteriochlorophyll component B870 to a form absorbing at 770 nm but had little effect on the “B800 + B850” system, causing only a reversible shift of the 850-nm band to 845 nm. Anti-reaction center serum, added to subcellular fractions from Rps. spheroides with 1% LDAO, precipitated reaction center chromoprotein unaccompanied by light harvesting bacteriocholorophyll. Other antisera precipitated light harvesting components and left the reaction center chromophores in solution. A major protein of apparent weight 45 kdaltons was found in relatively nonpigmented fractions from Rps. spheroides, associated with cell wall fragments. The 45-kdalton protein showed considerable interstrain variability, whereas the 9-kdalton and reaction center proteins appeared constant.

EFFECTS OF EXTRACTION AND REPLACEMENT OF UBIQUINONE UPON THE PHOTOCHEMICAL ACTIVITY OF REACTION CENTERS AND CHROMATOPHORES FROM RHODOPSEUDOMONAS SPHERIODES

1. Introduction Ubiquinone (UQ) has been implicated as a primary photochemical electron acceptor in bacterial photo- synthesis. Evidence for this has included light-induced optical absorbance changes suggesting disappearance of the oxidized form of UQ [ 1 ] and formation of the anionic semiquinone, UQ: [2,3], and an electron spin resonance signal appropriate for the semiquinone [4,.5] UQ also functions as a secondary electron acceptor coupled to the primary photochemical system [2,6,7]. Dried preparations from photosynthetic bacteria have been subjected to extraction with hydrocarbon solvents such as iso-octane in order to study the effects of depleting UQ [2,6-lo]. Such extraction removes the component of UQ that functions as a secondary electron acceptor, and this function can be restored by adding back UQ [2,9]. In these experiments the capacity for primary photochemistry, as manifested by the oxidation of bacteriochlorophyll (BChl), was not attenuated, casting some doubt on the role of UQ in the photochemistry. We show here, using chromatophores and reaction centers (RC’s) prepared from Rhodopseudomonas

Molar extinction coefficients and other properties of an improved reaction center preparation from Rhodopseudomonas viridis

Abstract Reaction centers have been purified from chromatophores of Rhodopseudomonas viridis by treatment with lauryl dimethyl amine oxide followed by hydroxyapatite chromatography and precipitation with ammonium sulfate. The absorption spectrum at low temperature shows bands at 531 and 543 nm, assigned to two molecules of bacteriopheophytin b. The 600 nm band of bacteriochlorophyll b is resolved at low temperature into components at 601 and 606.5 nm. At room temperature the light-induced difference spectrum shows a negative band centered at 615 nm, where the absorption spectrum shows only a weak shoulder adjacent to the 600 nm band. The fluorescence spectrum shows a band at 1000 nm and no fluorescence corresponding to the 830 nm absorption band. Two molecules of cytochrome 558 and three of cytochrome 552 accompany each reaction center. The differential extinction coefficient (reduced minus oxidized) of cytochrome 558 at 558 nm was estimated as 20 ± 2 mM−1 · cm−1 through a coupled reaction with equine cytochrome c. The extinction coefficient of reaction centers at 960 nm was determined to be 123 ± 25 mM−1 · cm−1 by measuring the light-induced bleaching of P-960 and the coupled oxidation of cytochrome 558. The corresponding extinction coefficient at 830 nm is 300 ± 65 mM−1 · cm−1. The absorbance ratio a 280 nm a 830 nm in our preparations was 2.1, and there was 190 kg protein per mol of reaction centers. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three major components of apparent molecular weights 31 000, 37 000 and 41 000.

Photochemical Electron Transport in Photosynthetic Reaction Centers from Rhodopseudomonas spheroides: I. Kinetics of the Oxidation and Reduction of P-870 As Affected by External Factors

Photosynthetic reaction centers from Rhodopseudomonas spheroides were prepared with the detergent lauryl dimethylamine oxide (LDAO). In contrast to reaction centers made with Triton X-100, these contained no cytochromes and little or no ubiquinone (UQ). The reduction of P-870, after its photochemical oxidation, was studied in these materials with the following results. In reaction centers made with Triton X-100, slow kinetic components (seconds to minutes) could be attributed to secondary electron acceptors or traps. In reaction centers made with LDAO the kinetics were predominantly fast (half-times, 100 msec or less); slower components could be introduced by adding UQ. Added UQ appeared to become bound to reaction centers made with LDAO, but the binding might have meant only that both components were trapped within detergent micelles. Ferricyanide could retard the reduction of oxidized P-870, apparently by capturing electrons from the reducing side of the photochemical system. Under conditions in which the participation of secondary electron acceptors seemed to have been eliminated, the recovery of P-870 was mainly by a first-order process with a half-time of about 60 msec at room temperature and 20-30 msec at about -80 degrees C and below. The transition with decreasing temperature suggested the presence of a mixed population, exhibiting both the 60 and 20 msec components, but variations in the absorption spectra with temperature did not suggest the presence of a mixed population. Absorption difference spectra in the ultraviolet were compatible with the idea that UQ added to reaction centers became reduced in the light.

Photochemical Electron Transport in Photosynthetic Reaction Centers: IV. Observations Related to the Reduced Photoproducts

The formation and dissipation of reduced photoproducts in photochemical reaction centers from Rhodopseudomonas spheroides has been studied in three independent ways: by direct chemical reduction, by photochemical reduction (illuminating reaction centers in the presence of weak reductants), and by adding electron acceptors to illuminated reaction centers to reverse the reduction. In every case the reduction is attended by the appearance of an absorption band at 450 nm and the bathochromic shift of a band centered at 305 nm. Both reduction and oxidation of reaction centers, and also photochemical oxidoreduction, cause bathochromic shifts of absorption bands identified with bacteriopheophytin (BPh), and hypsochromic shifts of bands of bacteriochlorophyll (BChl) (P-800 and, in the case of reduction, P-870). Reduction causes relatively large shifts of BPh and small shifts of BChl; the reverse is seen with oxidation and oxidoreduction. Addition of sodium dodecyl sulfate (SDS) to reaction centers suppresses the 450 nm absorption change but not the band shifts associated with BPh and BChl. Under some conditions the 450 nm change and the band shifts show different kinetics, with the kinetics of the band shifts matching those of a transient change in the yield of P-870 fluorescence. New data, on the efficiency of photo-bleaching of P-870 in reaction centers in which part of the P-870 has already been oxidized with ferricyanide, militate against the idea that part of the photochemical bleaching of P-870 is due to reduction of that pigment.