Paolo D. Gerola

LIGHT INTENSITY EFFECTS ON PIGMENT COMPOSITION AND ORGANISATION IN THE GREEN SULFUR BACTERIUM CHLOROBIUM TEPIDUM

We have investigated the changes in the pigment composition and organisation of the light-harvesting apparatus of the green sulfur bacterium Chlorobium tepidum growing under different light intensities. Cells grown at lower light intensities had lower exponential growth rates and increased amounts of the main light-harvesting pigments, bacteriochlorophyll c and carotenoids, on a cell protein basis. Absorption spectra of chlorosomes isolated from cells grown at low light intensities revealed a red-shift of up to 8 nm in the Qy band of bacteriochlorophyll c compared to chlorosomes from high light grown cells. A similar red-shift of up to 4 nm was also observed in the corresponding fluorescence emission peaks. HPLC analysis of pigment extracts showed a correlation between the red-shift and the content of the more alkylated BChl c homologs, which increased as light intensity for growth was lower. Furthermore, analysis of the carotenoid composition in chlorosomes re vealed a conspicuous change in the ratio between chlorobactene and 1′, 2′-dihydrochlorobactene, which dramatically decreased from 5 to 0.7 in light-limited cultures.

Partition zone penetration by chymotrypsin, and the localization of the chloroplast flavoprotein and Photosystem II

1. Chymotrypsin treatment of chloroplast membranes inactivates Photosystem II. The inactivation is higher when the activity is measured under low intensity actinic light, suggesting that primary photochemistry is preferentially inactivated. 2. Membrane stacking induced by Mg2+ protects Photosystem II against chymotrypsin inactivation. When the membranes are irreversible unstacked by brief treatment with trypsin, Mg2+ protection against chymotrypsin inactivation of Photosystem II is abolished. 3. The kinetics of inactivation by chymotrypsin of Photosystem II indicates that membrane stacking slows down, but does not prevent, the access of chymotrypsin to Photosystem II, which is mostly located within the partition zones. 4. It is concluded that a partition gap exists between stacked membranes of about 45 A, the size of the chymotrypsin molecule. 5. The kinetics of inhibition of the chloroplast flavoprotein, ferredoxin-NADP reductase, bt its specific antibody is not affected by membrane stacking. This indicates that this enzyme is located outside the partition zones.

Circular dichroism of green bacterial chlorosomes

Positive and negative bands in previously measured circular dichroism (CD) spectra of Chlorobium limicola chlorosomes appeared to be sign-reversed relative to those of Chloroflexus aurantiacus chlorosomes in the 740–750 nm spectral region where bacteriochlorophyll (BChl) c absorbs maximally. It was not clear, however, whether this difference was intrinsic to the chlorosomes or was due to differences in the procedures used to prepare them. We therefore repeated the CD measurements using chlorosomes isolated from both Cb. limicola f. thiosulfatophilum and Cf. aurantiacus using the method of Gerola and Olson (1986, Biochim. Biophys. Acta 848: 69–76). Contrary to the earlier results, both types of chlorosomes had very similar CD spectra, suggesting that both have similar arrangements of BChl c molecules. The previously reported difference between the CD spectra of Chlorobium and Chloroflexus chlorosomes is due to the instability of Chlorobium chlorosomes, which can undergo a hypsochromic shift in their near infrared absorption maximum accompanied by an apparent inversion in their near infrared CD spectrum during isolation. Treating isolated chlorosomes with the strong ionic detergent sodium dodecylsulfate, which removes BChl a, does not alter the arrangement of BChl c molecules in either Chloroflexus or Chlorobium chlorosomes, as indicated by the lack of an effect on their CD spectra.

A study on the lateral distribution of the plastoquinone pool with respect to Photosystem II in stacked and unstacked spinach chloroplasts

Abstract The quenching of Photosystem II (PS II) chlorophyll fluorescence by oxidised plastoquinone has been used in an attempt to determine their relative distribution in the partition zone and stroma-exposed thylakoid membranes. Thus, the PS II-plastoquinone interaction was determined in stacked (2.5 mM MgCl 2 ) and largely unstacked (0.25 mM MgCl 2 ) membranes. A method to correct for spillover or other quenching changes at the different MgCl 2 concentrations, which would compete with the plastoquinone-induced quenching, was devised utilising the quinone dibromothymoquinone. This compound is demonstrated to behave as an ideal (theoretically) PS II quencher at both high and low MgCl 2 concentrations, which indicates that it distributes itself homogeneously between partition zone and stroma-exposed membrane regions. In passing from the stacked to the unstacked configuration, the PS II-plastoquinone interaction decreases less than the PS II-dibromothymoquinone interaction. This is interpreted to mean that plastoquinone is present in both the partition zone and stroma-exposed membranes, with somewhat higher concentrations in the stroma-exposed membranes. Thus, plastoquinone is well placed to transport reducing equivalents from the partition zones to the stroma-exposed membranes.

Proton-induced grana formation in chloroplasts. Distribution of chlorophyll-protein complexes and Photosystem II photochemistry

Abstract Lowering the pH of the incubation medium to pH 5.4 leads to grana formation morphologically similar to that induced by metal cations. The same phenomenon is observed in EDTA-washed chloroplasts, indicating that it is not due in part to electrostatic ‘masking’ by residual cations associated with the membranes. Digitonin fractionation studies have indicated that the distribution of the major chlorophyll-protein complexes between granal and stromal membrane regions is similar at pH 5.4 in the absence of Mg 2+ , and at pH 7.4 in the presence of Mg 2+ . Chlorophyll fluorescence induction studies have indicated that the primary photochemistry of Photosystem II (PS II) is stimulated by lowering the pH to 5.4, just as it is upon metal cation addition at higher pH values. The failure to observe such an increase at pH 5.4 by measuring electron transport to ferricyanide is attributed to a combination of an inhibition by this pH of electron transport at a site after Q reduction and an increase in the number of PS II centres detached from the plastoquinone pool. We conclude that the stacked configuration of chloroplast membranes leads to increased PS II primary photochemistry, which is most simply explained in terms of a redistribution of excitation energy towards PS II.

Membrane phosphorylation leads to the partial detachment of the chlorophyll a/b protein from photosystem II

Abstract The hypothesis that the chlorophyll fluorescence decline due to membrane phosphorylation is caused principally by the detachment and removal of LHCP from the LHCP-PS II matrix is examined. It is demonstrated that when membranes are phosphorylated in the dark (a) the fluorescence decline is greater when excited by light enriched in wavelengths absorbed mainly by LHCP (475 nm) than when excited by light absorbed to a large extent also by the PS II complex (435 nm), (b) titration with different artificial quenchers of chlorophyll fluorescence is unchanged after the phosphorylation-induced fluorescence decline, and (c) the F v / F m ratio does not change after the phosphorylation-induced fluorescence decline. These data indicate that it is indeed principally LHCP that interacts with the quencher (PS I presumably). This interaction involves a small fraction of the total PS II-coupled LHCP, which becomes functionally detached from the LHCP-PS II matrix.

Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent Km for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.

Influence of protons on thylakoid membrane stacking

Abstract The effect of protons on thylakoid membranes was examined. On lowering the pH of the suspension medium to pH 5.4 in the presence of a low monovalent cation concentration and in the absence of divalent cations the broken chloroplasts changed from an unstacked to a stacked configuration, identical to that observed at pH 8 in the presence of 5 mM MgCl2. A brief trypsin treatment prevented the thylakoid membranes from forming grana stacks either at pH 8 in the presence of 5 mM MgCl2 or at pH 5.4. These observations indicate that the same protein site(s) is probably involved in membrane stacking caused either by protons or by metal cations at high pH.

Grana formation in chloroplasts may promote energy transfer between photosystem II units

The biological significance of grana formation in chloroplasts is of considerable interest. The suggestion that grana formation may be a means of achieving a high density of light harvesting assemblies [ 1 ] has not been corroborated (reviewed [2]). The quantum efficiency of reduction of electron acceptors added to isolated chloroplasts, stacked or unstacked, has been measured directly or indirectly [l--8]. However, at low light intensities and in the presence of an electron acceptor the primary electron acceptor of photosystem II, Q, is largely oxidised. Thus any effect of grana on energy transfer between photosystem II units would not have been detected [9,10]. Oxygen flash yield studies [9] demonstrated that MgClz (3 mM) or KC1 (100 mM) induces pronounced energy transfer between photosystem II units. The influence of cations on oxygen flash yield only became evident as Q became reduced. A similar conclusion was reached on the basis of fluorescence induction studies [ 111. As these cation concentrations are similar to those required for grana formation [12], it seemed possible that the two phenomena may be related. Here we have utilised the observation [2,13] that lowering the medium pH to 5.4 brings about the formation of grana which are morphologically indistinguishable from those induced by metal cations at neutral pH values. These grana seem also to be biochemically similar to those produced by metal cations as indicated by the chlorophylls/b ratios of digitonin fractions [ 141. We now demonstrate that incubation of chloroplasts at pH 5.4 strongly favours energy transfer between photosystem II units, and suggest that this may be due to grana formation.

STUDIES ON THE FRACTIONATION BY DIGITONIN OF CHLOROPLAST MEMBRANES

11. The basic action of digitonin was to separate the grana thyla- koids from the intergrana thylakoids [Z]. Thus the heavy fraction (10 000 X g pellet, enriched in photo- sytem II) consisted mostly of grana, while the light fraction (10 000 X g supernatant, enriched in photo- system I) consisted mostly of intergrana membranes. This method has been used as a quick and easy method to quantitate grana fo~ation [3-53. Grana formation and a normal digitonin fractionation pat- tern have been closely correlated [6] but have in the presence of 0.1 M ammonium acetate no membrane stacking to be observed with the electron micro- scope, although a normal digitonin fractionation pattern was observed [6]. This article reexamines the digitonin fractionation method to ascertain whether it is a reliable method for grana quantifica- tion. Our data show that, at least under certain con- ditions, factors other than the amount of grana strongly influence this fractionation procedure. 2.