Reconstitution of Photosynthetic Energy Conservation

Abstract

A preparation of reaction centers of photosystem I from spinach chloroplasts was incorporated into lipid vesicles by sonication. Incorporation was tested by chromatography on Sepharose-.IB, by comparison of the elution profiles of photosystem-I reaction center liposomes and of free reaction centers. In the presence of reduced N-methylphenazonium methosulfate the reaction center liposomes catalyzed net proton extrusion in the light, but at the same time showed light-induced quenching of 9-aminoacridine fluorescence, with similar extent and kinetics as known for chloroplasts. We conclude that we are dealing with two vesicle populations, one right side-out and one inside-out with respect to the orientation of the incorporated reaction center complex. Net proton movements are influenced by the nature of the cations present in the suspending medium, and the possibilities for effects of surface charge on these movements are discussed. During photosynthetic energy conservation in plants and bacteria light energy is primarily converted into a membrane potential by charge separation in the reaction centers followed by vectorial electron transport within asymmetrically organized membranes. The electron transport is coupled to transmembrane proton translocation, which leads to the formation of an electrochemical potential difference of protons. According to the chemiosmotic theory, this potential difference is the driving force for ATP synthesis [l -41. Therefore the minimal requirements for the reconstitution of photophosphorylation from chloroplast components in a liposomal model system might be: a reaction center chlorophyll-protein complex for the light-induced charge separation, positive inside the vesicle, an appropriate redox system for the formation of a transmembrane proton gradient, acidic also inside, and the coupling factor complex as the enzyme of ATP synthesis, accessible for substrates. Such a comparatively simple electron transport system is probably functioning in chloroplasts in the presence of reduced phenazine methosulfate, if electron flow between the two photosystems is inhibited [5].Abbreviations and Trivial Names. Phenazine methosulfate, Nmethylphenazonium methosulfate; aminoacridine, 9-aminoacridine; CF30PhzC(CN)Z, carbonylcyanide p-trifluoromethoxyphenylhydrazone, also known as FCCP; Tricine, N-[2-hydroxy-l,lbis(hydroxymethyl)ethyl]glycine; P700, reaction center chlorophyll of photosystem I.Reaction center complexes of higher plants and bacteria incorporated into phospholipid membranes have been studied before, in relation to structural and functional aspects. Electron microscopy of freezefractured reaction-center liposomes revealed particlecontaining fracture f x e s in contrast to smooth fracture faces of pure liposomes, indicating that the reaction center is embedded in the lipid matrix [6,7,]. Transmembrane arrangement has not been demonstrated so far by electron microscopy. Proteoliposomes [S] and planar membranes [9] inlayed with reaction centers isolated from photosynthetic bacteria have ...

Analysis of light-induced transmembrane ion gradients and membrane potential in Photosystem I proteoliposomes

“…Complexes were suspended in Tricine buffer (20 mM Tricine-NaOH, 0.06% β-DM, pH 7.5) at a chlorophyll concentration of 0.15 mg/ml. PSI proteoliposomes were prepared according to the method described in [14], but with minor modifications. In brief, soybean phosphatidylcholine, cholesterol and phosphatidic acid (molar ratio 7:2:1)…”

Reconstitution of Photosynthetic Energy Conservation

“…This has been described in the preceding paper [7]. Unfortunately only the minor fraction of the vesicles contained the photosystem-I reaction center in the polarity leading to proton uptake, resembling the orientation in chloroplasts [7] (see Fig. 7 ) .…”

“…The green band from the sucrose gradient was collected, concentrated and stored in liquid N2 as described in the preceding paper [7].…”

“…And indeed, we succeeded in obtaining proton translocation through the membrane of illuminated vesicles from soybean phospholipids inlayed with photosystem-I reaction centers, in the presence of reduced phenazine methosulfate. This has been described in the preceding paper [7]. Unfortunately only the minor fraction of the vesicles contained the photosystem-I reaction center in the polarity leading to proton uptake, resembling the orientation in chloroplasts [7] (see Fig.…”

A Cytochrome f/b6 Complex of Five Polypeptides with Plastoquinol‐Plastocyanin‐Oxidoreductase Activity from Spinach Chloroplasts

“…At this stage of research it would be desirable to study the cytochrome complex in an isolated state, either in micellar form, in the presence of detergents, or even reincorporated into defined lipid vesicles. Furthermore, since we succeeded recently to incorporate photosystem I reaction centers into liposomes, and to reconstitute proton translocation with artificial redox compounds [17], it would be promising to include the cytochrome complex in such a reconstituted system. Purification procedures for cytochromef'from spinach and other sources , and for cytochrome hb [22,23] have been published.…”