To elucidate the molecular basis of the link between respiration and longevity, we have studied the organization of the respiratory chain of a wild-type strain and of two long-lived mutants of the filamentous fungus Podospora anserina. This established aging model is able to respire by either the standard or the alternative pathway. In the latter pathway, electrons are directly transferred from ubiquinol to the alternative oxidase and thus bypass complexes III and IV. We show that the cytochrome c oxidase pathway is organized according to the mammalian "respirasome" model (Schä gger, H., and Pfeiffer, K. (2000) EMBO J. 19, 1777-1783). In contrast, the alternative pathway is composed of distinct supercomplexes of complexes I and III (i.e. I 2 and I 2 III 2 ), which have not been described so far. Enzymatic analysis reveals distinct functional properties of complexes I and III belonging to either cytochrome c oxidase-or alternative oxidase-dependent pathways. By a gentle colorless-native PAGE, almost all of the ATP synthases from mitochondria respiring by either pathway were preserved in the dimeric state. Our data are of significance for the understanding of both respiratory pathways as well as lifespan control and aging.

Higher plant mitochondria have many unique features compared with their animal and fungal counterparts. This is to a large extent related to the close functional interdependence of mitochondria and chloroplasts, in which the two ATP-generating processes of oxidative phosphorylation and photosynthesis, respectively, take place. We show that digitonin treatment of mitochondria contaminated with chloroplasts from spinach (Spinacia oleracea) green leaves at two different buffer conditions, performed to solubilize oxidative phosphorylation supercomplexes, selectively extracts the mitochondrial membrane protein complexes and only low amounts of stroma thylakoid membrane proteins. By analysis of digitonin extracts from partially purified mitochondria of green leaves from spinach using blue and colorless native electrophoresis, we demonstrate for the first time that in green plant tissue a substantial proportion of the respiratory complex IV is assembled with complexes I and III into "respirasome"-like supercomplexes, previously observed in mammalian, fungal, and non-green plant mitochondria only. Thus, fundamental features of the supramolecular organization of the standard respiratory complexes I, III, and IV as a respirasome are conserved in all higher eukaryotes. Because the plant respiratory chain is highly branched possessing additional alternative enzymes, the functional implications of the occurrence of respiratory supercomplexes in plant mitochondria are discussed.

Photosystem II (PSII) is the membrane protein complex that catalyzes the photo-induced oxidation of water at a manganesecalcium active site. Light-dependent damage and repair occur in PSII under conditions of high light stress. The core reaction center complex is composed of the D1, D2, CP43, and CP47 intrinsic polypeptides. In this study, a new chromophore formed from the oxidative post-translational modification of tryptophan is identified in the CP43 subunit. Tandem mass spectrometry peptide sequencing is consistent with the oxidation of the CP43 tryptophan side chain, Trp-365, to produce N-formylkynurenine (NFK). Characterization with ultraviolet visible absorption and ultraviolet resonance Raman spectroscopy supports this assignment. An optical assay suggests that the yield of NFK increases 2-fold (2.2 ؎ 0.5) under high light illumination. A concomitant 2.4 ؎ 0.5-fold decrease is observed in the steady-state rate of oxygen evolution under the high light conditions. NFK is the product formed from reaction of tryptophan with singlet oxygen, which can be produced under high light stress in PSII. Reactive oxygen species reactions lead to oxidative damage of the reaction center, D1 protein turnover, and inhibition of electron transfer. Our results are consistent with a role for the CP43 NFK modification in photoinhibition.Oxygenic photosynthesis is the enzyme-catalyzed conversion of light energy to biochemical energy, and this process occurs in the membranes of plants, algae, and cyanobacteria. In oxygenic photosynthesis, photosystem II (PSII) 4 catalyzes the light-driven oxidation of water and reduction of plastoquinone. On the acceptor side of PSII, electrons are transferred sequentially to two quinone molecules, Q A and Q B (1). On the donor side, a Mn 4 Ca active site is the binding site for water and the site of oxygen production. Each monomer is composed of 20 protein subunits, chlorophylls, carotenoids, and redox-active plastoquinones (2, 3). Calcium and chloride are required for activity under physiological conditions (4). The chloride binding site has been assigned near the active site (2, 3).The D1, D2, CP43, and CP47 polypeptides form the intrinsic core complex of PSII. The D1 and D2 membrane spanning proteins bind the electron transfer cofactors active in water oxidation (2, 3). This central heterodimeric core is symmetrically flanked by the CP43 and CP47 proteins, which bind light-harvesting antennae chlorophyll (Chl) molecules (5). Each of these core polypeptides is composed of intrinsic membrane-spanning helices, as well as several hydrophilic loops that protrude into the interior lumen of the thylakoid membrane (2, 3). The lumenal loop regions of CP43 have been implicated as important in assembly and protection from photoinhibition (see Ref. and references therein).The active site of water oxidation, the Mn 4 Ca cluster, is located on the lumenal surface and is protected by three extrinsic polypeptides (6). In plants, these extrinsic proteins, the 18-kDa, 24-kDa, and psbO (or the 33-kDa, manganes...

The light reactions of oxygenic photosynthesis almost invariably take place in the thylakoid membranes, a highly specialized internal membrane system located in the stroma of chloroplasts and the cytoplasm of cyanobacteria. The only known exception is the primordial cyanobacterium Gloeobacter violaceus, which evolved before the appearance of thylakoids and harbors the photosynthetic complexes in the plasma membrane. Thus, studies on G. violaceus not only shed light on the evolutionary origin and the functional advantages of thylakoid membranes but also might include insights regarding thylakoid formation during chloroplast differentiation. Based on biochemical isolation and direct in vivo characterization, we report here structural and functional domains in the cytoplasmic membrane of a cyanobacterium. Although G. violaceus has no internal membranes, it does have localized domains with apparently specialized functions in its plasma membrane, in which both the photosynthetic and the respiratory complexes are concentrated. These bioenergetic domains can be visualized by confocal microscopy, and they can be isolated by a simple procedure. Proteomic analysis of these domains indicates their physiological function and suggests a protein sorting mechanism via interaction with membrane-intrinsic terpenoids. Based on these results, we propose specialized domains in the plasma membrane as evolutionary precursors of thylakoids.

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H+-ATP synthase is the dominant ATP production site in mitochondria and chloroplasts. So far, dimerization of ATP synthase has been observed only in mitochondria by biochemical and electron microscopic investigations. Although the physiological relevance remains still enigmatic, dimerization was proposed to be a unique feature of the mitochondrion [Biochim. Biophys. Acta 1555 (2002) 154]. It is hard to imagine, however, that closely related protein complexes of mitochondria and chloroplast should show such severe differences in structural organization. We present the first evidences for dimerization of chloroplast ATP synthases within the thylakoid membrane. By investigation of the thylakoid membrane of Chlamydomonas reinhardtii by blue-native polyacrylamide gel electrophoresis, dimerization of the chloroplast ATP synthase was detected. Chloroplast ATP synthase dimer dissociates into monomers upon incubation with vanadate or phosphate but not by incubation with molybdate, while the mitochondrial dimer is not affected by the incubation. This suggests a distinct dimerization mechanism for mitochondrial and chloroplast ATP synthase. Since vanadate and phosphate bind to the active sites, contact sites located on the hydrophilic CF1 part are suggested for the chloroplast ATP synthase dimer. As the degree of dimerization varies with phosphate concentration, dimerization might be a response to low phosphate concentrations.

Analysis of the membrane integral proteome is mainly dependent on the ability of protein separation. Blue-native polyacrylamide gel electrophoresis (BN-PAGE) is a technique capable of efficient membrane protein separation, so far mainly applied to the mitochondrial oxidative phosphorylation machinery. Applying BN-PAGE to the thylakoid membranes after mild solubilization with digitonin we succeeded in displaying the response of the green algae Chlamydomonas reinhardtii to altered culture conditions. In addition, by peptide mass fingerprinting and matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS) extremely hydrophobic subunits of the photosystem complexes with 5-11 transmembrane helices were identified, which could not be accessed by in-gel digestion in previous studies.