Stephan Berry

Endosymbiosis and the design of eukaryotic electron transport.

The bioenergetic organelles of eukaryotic cells, mitochondria and chloroplasts, are derived from endosymbiotic bacteria. Their electron transport chains (ETCs) resemble those of free-living bacteria, but were tailored for energy transformation within the host cell. Parallel evolutionary processes in mitochondria and chloroplasts include reductive as well as expansive events: On one hand, bacterial complexes were lost in eukaryotes with a concomitant loss of metabolic flexibility. On the other hand, new subunits have been added to the remaining bacterial complexes, new complexes have been introduced, and elaborate folding patterns of the thylakoid and mitochondrial inner membranes have emerged. Some bacterial pathways were reinvented independently by eukaryotes, such as parallel routes for quinol oxidation or the use of various anaerobic electron acceptors. Multicellular organization and ontogenetic cycles in eukaryotes gave rise to further modifications of the bioenergetic organelles. Besides mitochondria and chloroplasts, eukaryotes have ETCs in other membranes, such as the plasma membrane (PM) redox system, or the cytochrome P450 (CYP) system. These systems have fewer complexes and simpler branching patterns than those in energy-transforming organelles, and they are often adapted to non-bioenergetic functions such as detoxification or cellular defense.

Fluorescent probes for non-invasive bioenergetic studies of whole cyanobacterial cells

Fluorescent DeltapH and DeltaPsi indicators have been screened for the non-invasive monitoring of bioenergetic processes in whole cells of the cyanobacterium Synechocystis sp. PCC 6803. Acridine yellow and Acridine orange proved to be the best DeltapH indicators for the investigation of thylakoid and cytoplasmic membrane energization: While Acridine yellow indicated only cytosolic energization, Acridine orange showed signals from both the thylakoid lumen and the cytosol that could be separated kinetically. Both indicators were applied successfully to monitor cellular energetics, such as the interplay of linear and cyclic photosynthetic electron transport, osmotic adaptation and solute transport across the cytoplasmic membrane. In contrast, useful membrane potential indicators were more difficult to find, with Di-4-ANEPPS and Brilliant cresyl blue being the only promising candidates for further studies. Finally, Acridine yellow and Acridine orange could also be applied successfully for the thermophilic cyanobacterium Synechococcus elongatus. Different from Synechocystis sp. PCC 6803, where both respiration and ATP hydrolysis could be utilized for cytoplasmic membrane energization, proton extrusion at the cytoplasmic membrane in Synechococcus elongatus was preferentially driven by ATP hydrolysis.

Proton to electron stoichiometry in electron transport of spinach thylakoids

According to the concept of the Q-cycle, the H+/e- ratio of the electron transport chain of thylakoids can be raised from 2 to 3 by means of the rereduction of plastoquinone across the cytochrome b6f complex. In order to investigate the H+/e- ratio we compared stationary rates of electron transport and proton translocation in spinach thylakoids both in the presence of the artificial electron acceptor ferricyanide and in the presence of the natural acceptor system ferredoxin+NADP. The results may be summarised as follows: (1) a variability of the H+/e- ratio occurs with either acceptor. H+/e- ratios of 3 (or even higher in the case of the natural acceptor system, see below) are decreased towards 2 if strong light intensity and low membrane permeability are employed. Mechanistically this could be explained by proton channels connecting the plastoquinol binding site alternatively to the lumenal or stromal side of the cytochrome b6f complex, giving rise to a proton slip reaction at high transmembrane DeltapH. In this slip reaction protons are deposited on the stromal instead of the lumenal side. In addition to the pH effect there seems to be a contribution of the redox state of the plastoquinone pool to the control of proton translocation; switching over to stromal proton deposition is favoured when the reduced state of plastoquinone becomes dominant. (2) In the presence of NADP a competition of both NADP and oxygen for the electrons supplied by photosystem I takes place, inducing a general increase of the H+/e- ratios above the values obtained with ferricyanide. The implications with respect to the adjustment of a proper ATP/NADPH ratio for CO2 reduction are discussed.

Potassium uptake in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 mainly depends on a Ktr-like system encoded by slr1509 (ntpJ)

The molecular basis of potassium uptake in cyanobacteria has not been elucidated. However, genes known from other bacteria to encode potassium transporters can be identified in the genome of Synechocystis sp. strain PCC 6803. Mutants defective in kdpA and ntpJ were generated and characterized to address the role of the Kdp and KtrAB systems in this strain. KtrAB is crucial for K+ uptake, as the ΔntpJ mutant shows slowed growth, slowed potassium uptake kinetics, and increased salt sensitivity. The ΔkdpA mutant has the same phenotype as the wild type even at limiting potassium, but a ΔkdpAΔntpJ double mutant is not viable, indicating a role of Kdp for potassium uptake when the Ktr system is not functioning.

Adaptation to iron deficiency: a comparison between the cyanobacterium Synechococcus elongatus PCC 7942 wild-type and a DpsA-free mutant

To learn more about the adaptive response of Synechococcus elongatus PCC 7942 to iron starvation and the role of DpsA, presumably a protein protecting chromosomal DNA against oxidative damage, we performed a comparative analysis of S. elongatus PCC 7942 wild-type and a DpsA-free mutant, called K11. Relative to wild-type, the DpsA-free mutant had significantly higher amounts of phycocyanin and allophycocyanin, even upon iron limitation. While the Photosystem I activity in mutant K11 remained high under iron deficiency, the Photosystem II activity dropped severely with respect to wild-type. The DpsA content in wild-type was already fairly high under regular growth conditions and did not significantly increase under iron deficiency nor in the presence of 0.3 mM 2′2′-dipyridyl in iron-sufficient BG11 medium. Nevertheless, the absence of DpsA in K11 resulted in a significantly altered transcriptional/translational activity of genes known to be involved in adaptation to iron starvation. The amount of isiA/B transcript was about two-fold lower than in wild-type, resulting in a lower 77 K chlorophyll a fluorescence at 685 nm, implying a lower concentration of Photosystem I-IsiA supercomplexes. While in wild-type idiA, idiB, and irpA transcripts were highly up-regulated, hardly any were detectable in mutant K11 under iron limitation. The concentration of mapA transcript, however, was greatly increased in K11 compared to wild-type. Measurements of acridine yellow fluorescence with intact wild-type and K11 cells revealed that iron deficiency caused an increased contribution of cyclic electron transport to membrane energisation and ATP synthesis being in agreement with the formation of the Photosystem I-IsiA supercomplex. In addition, mutant K11 had a much higher respiratory activity compared to wild-type under iron limitation.

The Chemical Basis of Membrane Bioenergetics

Abstract. All organisms rely on chemiosmotic membrane systems for energy transduction; the great variety of participating proteins and pathways can be reduced to a few universal principles of operation. This chemical basis of bioenergetics is reviewed with respect to the origin and early evolution of life. For several of the cofactors which play important roles in bioenergetic reactions, plausible prebiotic sources have been proposed, and it seems likely that these cofactors were present before elaborate protein structures. In particular, the hydrophobic quinones require only a membrane-enclosed compartment to yield a minimum chemiosmotic system, since they can couple electron transport and proton translocation in a simple way. It is argued that the central features of modern bioenergetics, such as the coupling of redox reactions and ion translocation at the cytoplasmic membrane, probably are ancient features which arose early during the process of biogenesis. The notion of a thermophile root of the universal phylogenetic tree has been discussed controversially, nevertheless, thermophiles are interesting model organisms for reconstructing the origin of chemiosmotic systems, since they are often acidophiles and anaerobic respirers exploiting iron–sulfur chemistry. This perspective can help to explain the prominent role of iron–sulfur proteins in extant biochemistry as well as the origin of both respiration and proton extrusion within the context of a possible origin of life in the vicinity of hot vents.

Kinetic modeling of the photosynthetic electron transport chain.

A simulation model of the photosynthetic electron transport chain operating under steady state conditions is presented. The model enables the calculation of (1) the rates of electron transport and transmembrane proton translocation, (2) the proton/electron stoichiometry, (3) the number of electrons stored in the different redox centers and (4) the stationary transmembrane pH difference. Light intensity and proton permeability of the thylakoid membrane are varied in order to compare the predictions of the model with experimental data. The routes of electron transport and proton translocation are simulated by two coupled arithmetic loops. The first one represents the sequence of reaction steps making up the linear electron transport chain and the Q-cycle. This loop yields the electron flow rate and the proton/electron ratio. The second loop balances the H+ fluxes and yields the internal H+ concentration. The bifurcation of the electron transport pathways at the stage of plastoquinol oxidation is obligatory. The first electron enters always the linear branch and is transferred to photosystem I. The electron of the remaining semiquinone can enter the Q-cycle or, alternatively, the semiquinone can be lost from the cytochrome b6f complex. The competition between these two reactions explains the experimentally observed variability of the proton/electron ratio. We also investigated additional model variants, where the variation of the proton/electron stoichiometry is attributed to other loss reactions within the cytochrome b6f complex. However, the semiquinone detachment seems to be the best candidate for a satisfactory description of the experimental data. Additional calculations were done in order to assess the effects of the movement of the Rieske protein on linear electron transport; it was found that this conformational change does not limit the electron transport rate, if it occurs with a time constant of at least 1000 s(-1).

On the Problem of Laws in Nature and History: A Comparison

In the philosophy of science there has traditionally been a tendency to regard physics as the incarnation of science per se. Consequently, the status of other disciplines has been evaluated according to their ability to produce laws resembling those of physics. This view has yielded a considerable bias in the discussion of historical laws. Philosophers as well as historians have tended to discuss such laws mostly with reference to the situation in physics; this often led to either one of two conclusions, namely that (1) history is epistemologically completely separated from natural science because it does not have universal laws, or that (2) the ultimate goal of the study of history must be the formulation of such universal laws. I maintain that neither conclusion is necessary. To substantiate this position, I discuss several aspects of natural laws. One aspect that is often neglected is that there are many kinds of statistical laws in nature; there is no close link between laws and determinism. Moreover, natural systems exist that have a history, that is, systems that are, like human history, shaped by irreversible, singular events. One important case is biological evolution; accordingly I discuss the relation between evolutionary theory and historiography. However, since we are part of the living world, and in addition to considering the methodological similarities between the two fields, one could also ask whether the laws of evolution are of direct relevance for understanding our history. This issue of history as evolution is investigated in detail in the final section of the paper.

Purification and characterisation of photosystem I from the unusual cyanobacteriumGloeobacter violaceus

Photosystem I from the highly unusual primitive cyanobacterium Gloeobacter violaceus was purified. The isolated Photosystem I complexes have the same subunit composition as complexes from other cyanobacteria as determined by high resolution gel electrophoresis and immunoblotting. They exist both in a trimeric and in a monomeric form as shown by gel filtration and electron microscopy. Image analysis of single particles shows that the size and shape of the particles closely resembles other cyanobacterial Photosystem I particles. Flash induced absorption difference spectra show a classical P700 shape with kinetics and relaxation times similar to Photosystem I from Synechocystis, but its amplitude indicates a much bigger antenna size, resembling the spectra of higher plants rather than those of other cyanobacteria. 77 K fluorescence emission spectra lack the typical long wavelength emission peaks of cyanobacterial Photosystem I. In addition, the preparation contains novel polypeptides not related to known Photosystem I subunits. Immunoblotting shows the possible existence of a membrane intrinsic light-harvesting protein related to LHC proteins of Chromophytes. Moreover, electron microscopy reveals a novel T-shaped complex that has not been observed so far.