Sascha Rexroth

Supramolecular Organization of Cytochrome c Oxidase- and Alternative Oxidase-dependent Respiratory Chains in the Filamentous Fungus Podospora anserina

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 (Schägger, 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. I2 and I2III2), 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.

The Plasma Membrane of the Cyanobacterium Gloeobacter violaceus Contains Segregated Bioenergetic Domains

Gloeobacter violaceus, the most primordial of known organisms performing oxygenic photosynthesis, evolved before the evolutionary appearance of thylakoid membranes. This study demonstrates the presence of bioenergetic domains in its cytoplasmic membrane. The formation of these membrane domains might constitute evolutionary precursors of the thylakoid membrane. 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.

Localization of cytochrome b6f complexes implies an incomplete respiratory chain in cytoplasmic membranes of the cyanobacterium Synechocystis sp. PCC 6803.

The cytochrome b(6)f complex is an integral part of the photosynthetic and respiratory electron transfer chain of oxygenic photosynthetic bacteria. The core of this complex is composed of four subunits, cytochrome b, cytochrome f, subunit IV and the Rieske protein (PetC). In this study deletion mutants of all three petC genes of Synechocystis sp. PCC 6803 were constructed to investigate their localization, involvement in electron transfer, respiration and photohydrogen evolution. Immunoblots revealed that PetC1, PetC2, and all other core subunits were exclusively localized in the thylakoids, while the third Rieske protein (PetC3) was the only subunit found in the cytoplasmic membrane. Deletion of petC3 and both of the quinol oxidases failed to elicit a change in respiration rate, when compared to the respective oxidase mutant. This supports a different function of PetC3 other than respiratory electron transfer. We conclude that the cytoplasmic membrane of Synechocystis lacks both a cytochrome c oxidase and the cytochrome b(6)f complex and present a model for the major electron transfer pathways in the two membranes of Synechocystis. In this model there is no proton pumping electron transfer complex in the cytoplasmic membrane. Cyclic electron transfer was impaired in all petC1 mutants. Nonetheless, hydrogenase activity and photohydrogen evolution of all mutants were similar to wild type cells. A reduced linear electron transfer and an increased quinol oxidase activity seem to counteract an increased hydrogen evolution in this case. This adds further support to the close interplay between the cytochrome bd oxidase and the bidirectional hydrogenase.

From protons to OXPHOS supercomplexes and Alzheimer's disease: Structure–dynamics–function relationships of energy-transducing membranes

By the elucidation of high-resolution structures the view of the bioenergetic processes has become more precise. But in the face of these fundamental advances, many problems are still unresolved. We have examined a variety of aspects of energy-transducing membranes from large protein complexes down to the level of protons and functional relevant picosecond protein dynamics. Based on the central role of the ATP synthase for supplying the biological fuel ATP, one main emphasis was put on this protein complex from both chloroplast and mitochondria. In particular the stoichiometry of protons required for the synthesis of one ATP molecule and the supramolecular organisation of ATP synthases were examined. Since formation of supercomplexes also concerns other complexes of the respiratory chain, our work was directed to unravel this kind of organisation, e.g. of the OXPHOS supercomplex I(1)III(2)IV(1), in terms of structure and function. Not only the large protein complexes or supercomplexes work as key players for biological energy conversion, but also small components as quinones which facilitate the transfer of electrons and protons. Therefore, their location in the membrane profile was determined by neutron diffraction. Physico-chemical features of the path of protons from the generators of the electrochemical gradient to the ATP synthase, as well as of their interaction with the membrane surface, could be elucidated by time-resolved absorption spectroscopy in combination with optical pH indicators. Diseases such as Alzheimers dementia (AD) are triggered by perturbation of membranes and bioenergetics as demonstrated by our neutron scattering studies.

Structural and functional alterations of cyanobacterial phycobilisomes induced by high-light stress

Exposure of cyanobacterial or red algal cells to high light has been proposed to lead to excitonic decoupling of the phycobilisome antennae (PBSs) from the reaction centers. Here we show that excitonic decoupling of PBSs of Synechocystis sp. PCC 6803 is induced by strong light at wavelengths that excite either phycobilin or chlorophyll pigments. We further show that decoupling is generally followed by disassembly of the antenna complexes and/or their detachment from the thylakoid membrane. Based on a previously proposed mechanism, we suggest that local heat transients generated in the PBSs by non-radiative energy dissipation lead to alterations in thermo-labile elements, likely in certain rod and core linker polypeptides. These alterations disrupt the transfer of excitation energy within and from the PBSs and destabilize the antenna complexes and/or promote their dissociation from the reaction centers and from the thylakoid membranes. Possible implications of the aforementioned alterations to adaptation of cyanobacteria to light and other environmental stresses are discussed.

N-formylkynurenine as a marker of high light stress in photosynthesis.

Photosystem II (PSII) is the membrane protein complex that catalyzes the photo-induced oxidation of water at a manganese-calcium 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.

Reactive oxygen species target specific tryptophan site in the mitochondrial ATP synthase.

The release of reactive oxygen species (ROS) as side products of aerobic metabolism in the mitochondria is an unavoidable consequence. As the capacity of organisms to deal with this exposure declines with age, accumulation of molecular damage caused by ROS has been defined as one of the central events during the ageing process in biological systems as well as in numerous diseases such as Alzheimers and Parkinsons Dementia. In the filamentous fungus Podospora anserina, an ageing model with a clear defined mitochondrial etiology of ageing, in addition to the mitochondrial aconitase the ATP synthase alpha subunit was defined recently as a hot spot for oxidative modifications induced by ROS. In this report we show, that this reactivity is not randomly distributed over the ATP Synthase, but is channeled to a single tryptophan residue 503. This residue serves as an intra-molecular quencher for oxidative species and might also be involved in the metabolic perception of oxidative stress or regulation of enzyme activity. A putative metal binding site in the proximity of this tryptophan residue appears to be crucial for the molecular mechanism for the selective targeting of oxidative damage.

Unique Properties vs. Common Themes: The Atypical Cyanobacterium Gloeobacter violaceus PCC 7421 is Capable of State Transitions and Blue-Light-Induced Fluorescence Quenching

The atypical unicellular cyanobacterium Gloeobacter violaceus PCC 7421, which diverged very early during the evolution of cyanobacteria, can be regarded as a key organism for understanding many structural, functional, regulatory and evolutionary aspects of oxygenic photosynthesis. In the present work, the performance of two basic photosynthetic adaptation/protection mechanisms, common to all other oxygenic photoautrophs, had been challenged in this ancient cyanobacterium which lacks thylakoid membranes: state transitions and non-photochemical fluorescence quenching. Both low temperature fluorescence spectra and room temperature fluorescence transients show that G. violaceus is capable of performing state transitions similar to evolutionarily more recent cyanobacteria, being in state 2 in darkness and in state 1 upon illumination by weak blue or far-red light. Compared with state 2, variable fluorescence yield in state 1 is strongly enhanced (almost 80%), while the functional absorption cross-section of PSII is only increased by 8%. In contrast to weak blue light, which enhances fluorescence yield via state 1 formation, strong blue light reversibly quenches Chl fluorescence in G. violaceus. This strongly suggests regulated heat dissipation which is triggered by the orange carotenoid protein whose presence was directly proven by immunoblotting and mass spectrometry in this primordial cyanobacterium. The results are discussed in the framework of cyanobacterial evolution.

OXPHOS Supercomplexes Respiration and Life-Span Control in the Aging Model Podospora anserina

Abstract:  Recent biochemical evidence has indicated the existence of respiratory supercomplexes as well as ATP synthase oligomers in the inner mitochondrial membrane of different eukaryotes. 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 aging model is able to respire by either the standard or the alternative pathway. In the latter, electrons are directly transferred from ubiquinol to the alternative oxidase (AOX) and thus bypass complexes III and IV. We showed that the two pathways are composed of distinct respiratory supercomplexes. These data are of significance for the understanding of both respiratory pathways as well as of life‐span control and aging.

Direct approach for bioprocess optimization in a continuous flat-bed photobioreactor system.

Application of photosynthetic micro-organisms, such as cyanobacteria and green algae, for the carbon neutral energy production raises the need for cost-efficient photobiological processes. Optimization of these processes requires permanent control of many independent and mutably dependent parameters, for which a continuous cultivation approach has significant advantages. As central factors like the cell density can be kept constant by turbidostatic control, light intensity and iron content with its strong impact on productivity can be optimized. Both are key parameters due to their strong dependence on photosynthetic activity. Here we introduce an engineered low-cost 5 L flat-plate photobioreactor in combination with a simple and efficient optimization procedure for continuous photo-cultivation of microalgae. Based on direct determination of the growth rate at constant cell densities and the continuous measurement of O₂ evolution, stress conditions and their effect on the photosynthetic productivity can be directly observed.