Hans-Erik Åkerlund

Reconstitution of photosynthetic water splitting in inside-out thylakoid vesicles and identification of a participating polypeptide☆

Inside-out spinach thylakoid vesicles have been used for inhibition and reconstitution of photosynthetic water splitting. Washing inside-out vesicles with a buffer containing 250 mM NaCl inhibited 75% of the Photosystem II reduction of phenyl-p-benzoquinone. In contrast, the same treatment of right-side-out vesicles gave quite a small inhibition. The site of inhibition was shown by fluorescence induction to be located at the water-splitting side of P-680. The salt-induced inhibition was accompanied by the release of two polypeptides (23 and 16 kDa) from the inner thylakoid surface. Readdition of the salt-washed supernatant or a crude chloroplast extract to the washed inside-out vesicles at low ionic strength gave a 2.7-fold stimulation of the water-splitting activity, thereby restoring about 60% of the activity lost by salt washing. This stimulation was abolished by proteolysis of the reconstituting fractions. Restoration of the water-splitting reaction was accompanied by rebinding of the 23 and 16 kDa polypeptides to the inside-out vesicles. After polypeptide purification by ion-exchange chromatography it was shown that the 23kDa polypeptide was responsible for the restorative effect. These observations provide strong evidence that the 23 kDa polypeptide, electrostatically bound to the inner thylakoid surface, is involved in the photosynthetic water-splitting reaction.

Localization of a 34 000 and a 23 000 Mr polypeptide to the lumenal side of the thylakoid membrane

Several attempts have been made to localize different components across the thylakoid membrane. The methods used include treatments with membrane-impermeable agents such as antibodies [ 11, chemical modifiers [ 2,3] and proteolytic enzymes [3]. This has given valuable information about the outer surface of the thylakoid membrane and has revealed that components like ferredoxin, ferredoxin-NADP-reductase, the coupling factor and the light-harvesting complex ‘are all accessible from the outside. Information about the inner thylakoid surface is poor however and the localization of components on this side has often been deduced from indirect measurements. A direct study of the inner side has become possible after the isolation of insideout thylakoids [4-91. In [IO] trypsination of insideout thylakoid vesicles was used to demonstrate that at least a part of the water-splitting system is exposed to the lumen. Here, inside-out thylakoid vesicles have been treated with trypsin or alkaline-Tris. The subsequent changes in the polypeptide pattern were followed by SDS-PAGE.

Regulation of violaxanthin de-epoxidase activity by pH and ascorbate concentration

The activity of violaxanthin de-epoxidase has been studied both in isolated thylakoids and after partial purification, as a function of pH and ascorbate concentration. We demonstrate that violaxanthin de-epoxidase has a Km for ascorbate that is strongly dependent on pH, with values of 10, 2.5, 1.0 and 0.3 mM at pH 6.0, 5.5, 5.0 and 4.5, respectively. These values can be expressed as a single Km±0.1±0.02 mM for the acid form of ascorbate. Release of the protein from the thylakoids by sonication was also found to be strongly pH dependent with a cooperativity of 4 with respect to protons and with an inflexion point at pH 6.7. These results can explain some of the discrepancies reported in the literature and provide a more consistent view of zeaxanthin formation in vivo.

Inside-out membrane vesicles isolated from spinach thylakoids

Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The sepration was obtained by partitionin an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: (1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl-p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; (4) concave EF and convex PF freeze-fracture faces.

Isolation of Photosystem II enriched membrane vesicles from spinach chloroplasts by phase partition

Partition in an aqueous Dextran-polyethylene glycol two-phase system has been used for the separation of chloroplast membrane vesicles obtained by press treatment of a grana-enriched fraction after unstacking in a low salt buffer. The fractions obtained were analysed with respect to chlorophyll, photochemical activities and ultrastructural charasteristics. The results reveal that the material partitioning to the Dextran-rich bottom phase consisted of large membrane vesicles possessing mainly Photosystem II properties with very low contribution from Photosystem I. Measurements of the H2O to phenyl-p-benzoquinone and ascorbate-Cl2Ind to NADP+ electron transport rates indicate a ratio of around six between Photosystem II and I. The total fractionation procedure could be completed within 2-3 h with high recovery of both the Photosystem II water-splitting activity and the Photosystem I reduction of NADP+. These data demonstrate that press treatment of low-salt destabilized grana membranes yields a population of highly Photosystem-II enriched membrane vesicles which can be discriminated by the phase system. We suggest that such membrane vesicles originate from large regions in the native grana membrane which contain virtually only Photosystem II.

Separation of subchloroplast membrane particles by counter-current distribution

Counter-current distribution in an aqueous Dextran-polyethylene glycol two-phase system has been used to fractionate membrane fragments obtained by press treatment of Class II chloroplasts. By the counter-current distribution technique membrane particles are separated according to their surface properties such as charge and hydrophobicity. The fractions obtained were analysed with respect to photochemical activities, chlorophyll and P-700 contents. The Photosystem II enrichment after counter-current distribution was better than that obtained by differential centrifugation of the disrupted chloroplasts. However, the best separation of Photosystem I and II enriched particles could be achieved if differential centrifugation was combined with the counter-current distribution technique. Each centrifugal fraction could be further separated into Photosystems I and II enriched fractions since the Photosystem II particles preferred the dextran-rich bottom phase while the Photosystem I particles preferred the polyethylene glycol-rich top phase. By this procedure it was possible, without the use of detergents, to obtain vesicles which were more enriched in Photosystem II as compared to intact grana stacks. The partition behaviour of undisrupted Class II chloroplasts and the Photosystem I centrifugal fraction was the same. This similarity indicated that the membrane which is exposed to the surrounding polymers by the Class II chloroplasts is the Photosystem I rich membrane of the stroma lamellae.

Immunological studies on the organization of proteins in photosynthetic oxygen evolution

Abstract Studies on inside-out thylakoid vesicles and several Photosystem-II particles have suggested the involvement of three proteins of 33, 23 and 16 kDa in photosynthetic oxygen evolution. In this study, monospecific antibodies were raised against the purified 33, 23 and 16 kDa proteins. The antibodies were used to investigate the organization and function of these proteins in the oxygen-evolving complex. Quantification of the 33, 23 and 16 kDa proteins by rocket immunoelectrophoresis revealed one copy of each polypeptide per some 200 chlorophylls in unfractionated thylakoids. Isolated inside-out thylakoids, derived from the grana partitions, showed 6–8 times more of the 33, 23 and 16 kDa proteins, on a chlorophyll basis, compared to the stroma lamellae vesicles. Agglutination studies revealed that the proteins are exposed on the lumenal side of the thylakoid membrane. An obligatory role for the 23 kDa protein in the photosynthetic water oxidation is suggested from the close correlation obtained between the inhibition of oxygen evolution and the release of this protein as caused by washing the inside-out thylakoids with increasing concentrations of NaCl. For the 16 kDa protein no such correlation was obtained. Rebinding experiments, using both salt-washed and alkaline Tris-washed inside-out thylakoids revealed that the 33 kDa protein was required for the binding of the 23 kDa protein, which in turn enhanced the binding of the 16 kDa protein. It is concluded that the three proteins are closely organized as a complex at the inner thylakoid surface in association with membrane spanning proteins of Photosystem II.

Studies on the polypeptide composition of the cyanobacterial oxygen-evolving complex

Abstract Various approaches have been used to investigate the polypeptides required for oxygen evolution in cyanobacteria, in particular the thermophile Phormidium laminosum . Antibodies against the extrinsic 33 kDa protein from spinach Photosystem II cross-reacted clearly in immunoblotting experiments with a corresponding polypeptide in isolated thylakoids and Photosystem II particles from P. laminosum and with whole-cell homogenates of three species of cyanobacteria ( Phormidium laminosum, Synechococcus leopoliensis and Anabaena variabilis ). In contrast, no cyanobacterial proteins reacted with antibodies against the 23 and 16 kDa proteins of spinach Photosystem II. The lack of cross-reactivity and the absence of these polypeptides from highly active Photosystem II particles of Phormidium laminosum strongly suggest that cyanobacteria do not contain polypeptides corresponding to these two chloroplast proteins. Treatment of P. laminosum Photosystem II particles with 0.8 M alkaline Tris, 1 M NaCl, CaCl 2 or MgCl 2 inhibited O 2 evolution, and quantitatively removed a 9 kDa polypeptide from the particles. None of these treatments removed comparable amounts of the 33 kDa polypeptide, and only Tris treatment removed manganese. The release of the 9 kDa polypeptide upon NaCl treatment correlated well with the deactivation at the donor side of Photosystem II. A direct connection between the 33 kDa polypeptide and O 2 evolution was established by the finding that trypsin treatment digested this polypeptide and inhibited O 2 evolution in parallel.

Differential phosphorylation of the light-harvesting chlorophyll—protein complex in appressed and non-appressed regions of the thylakoid membrane

It is now established that the light‐harvesting chlorophyll—protein complex (LHCP) of chloroplasts becomes phosphorylated in the light. In this study subfractionation of phosphorylated intact chloroplasts has been carried out to compare the phosphorylation of LHCP in non‐appressed and appressed thylakoid regions. The results show around 10‐times higher relative phosphorylation in the non‐appressed regions than in the appressed ones. Since the non‐appressed thylakoids also contain almost all photosystem 1, this region is likely to be the site for energy transfer from LHCP to photosystem 1 under phosphorylated conditions.

H2O2 accessibility to the photosystem II donor side in protein-depleted inside-out thylakoids measured as flash-induced oxygen production

Abstract The oxygen yield pattern from Photosystem II-enriched inside-out vesicles depleted of the 16 and 23 kDa polypeptides was studied. Two changes were observed. Firstly, there was as expected a decrease in the average amplitude due to the overall inhibition of oxygen-evolving capacity. Secondly, a signal was observed already at the first flash. This latter change in oscillation pattern was found to be caused by H 2 O 2 and weakly bound manganese present in the material. Thus, catalase, EDTA and high salt concentrations inhibited the signal on the first flash, while addition of H 2 O 2 or MnCl 2 increased the signal. The interpretation of these results is that removal of the 16 and 23 kDa proteins modifies the structure of the oxygen-evolving complex in such a way that it exposes the water-splitting site and makes it possible for H 2 O 2 to act as an electron donor to Photosystem II even at low concentrations.