Studies on the structure-function relationship of the cytochrome bc1 complex from Paracoccus denitrificans

Der Cytochrom bc1 Komplex spielt in der mitochondrialen Atmungskette eine zentrale Rolle, er ist am Aufbau des Protonengradienten über die innere Mitochondrien-Membran beteiligt. Die Funktionsweise dieses integralen Membranproteinkomplexes ist trotz mehrerer gelöster Strukturen noch nicht vollständig verstanden. Im Rahmen dieser Arbeit wurde der Komplex aus P. denitrificans untersucht und dabei mehrere Beiträge zu Struktur und Funktion dieses bakteriellen Modellsystems geleistet, wie z.B. seinem Oligomerenzustand in Detergenz-gelöster Form und zur Frage der Monomer:Monomer-Interaktion. Zur Strukturaufklärung des Cytochrom bc1 Komplexes aus P. denitrificans wurde ein chimärer Komplex zur Kristallisation eingesetzt, der in der Lage ist ein, Antikörper-Fragment zu binden, das bereits mit Erfolg zur Strukturbestimmung des Hefe-Komplexes verwendet wurde. Diese Experimente führten vermutlich wegen mangelnder Proteinstabilität nicht zum gewünschten Ergebnis. Eine zweite Mutante, bei der eine stark saure Domäne des Cyt c1 deletiert vorliegt (Δac Cytochrom bc1 Komplex), konnte jedoch erfolgreich kristallisiert und Diffraktionsmuster bis etwa 3,5 Ǻ erhalten werden. Die Kristalle weisen derzeit noch Inhomogenitäten, wahrscheinlich durch den Einfrierprozess, auf und werden gegenwärtig weiter optimiert. Im Rahmen dieser Arbeit konnte der lange diskutierte Oligomerenzustand des Cytochrom bc1 Komplexes aus P. denitrificans in solubilisiertem Zustand als Tetramer, beziehungsweise unter Einbeziehung struktureller und mechanistischer Daten und Überlegungen als Dimer eines Dimers, geklärt werden. Dies erfolgte durch eine für Membranproteine neue Form der Massenspektrometrie, die als LILBID-MS bezeichnet wird. Diese Daten konnten die bisher vorläufigen Beobachtungen aus BN-Gelelektrophorese und Gelfiltrationsversuchen eindeutig und unabhängig bestätigen. Darüber hinaus sollten noch Beiträge zur funktionellen Monomer:Monomer-Wechselwirkung geliefert werden. Hierfür wurde zunächst ein P. denitrificans Stamm erzeugt, der stabil zwei unterschiedliche fbc-Operons trägt und exprimiert: eine Wildtyp-Version mit Strep-tag und eine mit einem His-tag und einer inaktivierenden Mutation im Cyt b. Aus diesem Stamm sollte durch eine Tandem-Aufreinigung ein gemischter Cytochrom bc1 Komplex isoliert werden. Dies gelang nicht, wie in Westernblots, turnover und pre-steady-state Aktivitätsuntersuchungen gezeigt wurde, da sich der Komplex als Tetramer erwies und damit eine eindeutige Aufreinigung nicht möglich war. Die Lösung für dieses Problem liegt im Δac Cytochrom bc1 Komplex, der wie LILBID-MS Ergebnisse zeigten, lediglich als Dimer vorliegt; dazu müssen zukünftig die Affinitäts-tags und die inaktivierende Mutation auf diesen Komplex übertragen werden. Der zweite Beitrag zur Funktionsuntersuchung wurde anhand von Mutanten konservierter saurer Aminosäuren und von Tyrosinen im und um das QP-Zentrum geliefert, mit anschließenden Aktivitätsuntersuchungen, Inhibitor-Bindungstudien mit Stigmatellin und elektrochemisch-induzierten Redox-FTIR-Differenzspektren. Die Mutanten E295Q, E81Q und Y297Q zeigten eine verringerte Sensitivität für Stigmatellin. Die FTIR-Differenzspektren belegen, dass die Mutation der Positionen E295 und D278 die Signale für protonierte Seitengruppen verschieben. Die Mutation der Seitengruppen Y302, Y297, E81 und E295 beeinflussen direkt das oxidierte Chinon und das Proteinrückgrat. Die Bedeutung der lange diskutierten Seitengruppe E295 ließ sich als direkter Interaktionspartner mit dem Hydrochinon bestätigen. Die wichtige Rolle der Seitengruppen in Positionen E295 und Y302 konnte bestätigt werden. Darüber hinaus wurden die Seitengruppen D278 und E81 als wesentliche Wechselwirkungspartner für die Hydrochinon-Oxidation identifiziert. Die Seitengruppen des QP-Zentrums unterliegen durch das Wasserstoffbrückennetzwerk starken Wechselwirkungen, wodurch die Bindungstasche eine gewisse Toleranz gegenüber Veränderungen zeigt.The cytochrome bc1 complex is the central enzyme in the mitochondrial respiratory chain. It contributes to the proton gradient across the inner mitochondrial membrane. The mechanism of this integral membrane complex is not yet fully understood despite 3-D structures available. In this study the complex of P. denitrificans was investigated by different approaches with respect to structure and function, for example the oligomerisation state of the complex solubilized in detergent and the question of monomer:monomer interaction. The soil bacterium P. denitrificans is an established model organism for the mitochondrial respiratory chain due to its homologous complexes but simpler subunit composition. For structural studies of the complex from P. denitrificans a chimeric mutant was used. This complex was modified to bind an antibody fragment which was already successfully used for the structure determination of the yeast complex. These experiments, however,, failed, possibly due to protein instability. A second mutant, which lacks a highly acidic domain in the Cyt c1 (Δac Cytochrom bc1 complex), was crystallized and showed a diffraction pattern of around 3.5 Ǻ. For a higher resolution, crystals were not sufficiently homogenous possibly due to the freezing process. The long discussed detergent-solubilized oligomerization state of the complex is that of a tetramer, as shown by LILBID-MS, newly introduced to the field of membrane proteins. Based on structural and mechanistical results, complex-III should be considered a dimer of dimers. With these results the findings from BN-PAGE and gel filtration experiments were validated independently. Further investigations on monomer:monomer interactions were started. A P. denitrificans strain was constructed which stably expresses two different fbc-operons: a strep-tagged wild type version and a His-tagged version with an inactivating mutation in the cytochrome b gene to produce a mixed dimer amenable to tandem-affinity purification. As shown by Westernblots, turnover and pre-steady-state kinetics, it was not possible to isolate a pure mixed dimer, due to its tetrameric state. The solution for this problem lies in the Δac cytochrome bc1 complex mutant which is a dimer as shown by LILBID-MS. The described genetic background has to be transferred to this strain. Another contribution to the functional investigation was made using mutants of conserved acidic and tyrosine residues in and around the QP-site of the complex. The mutants were characterized by turnover and inhibitor binding studies with stigmatellin. Furthermore electrochemically induced FT-IR difference spectra were recorded. Mutants E295Q, E81Q and Y297Q showed lowered sensitivity for stigmatellin, and mutations in the positions E295 and D278 shifted the signal for protonated residues. Mutations in positions Y302, Y297, E81 and E295 showed a direct influence on the oxidized quinone and the protein backbone. The long discussed side chain E295 was verified to directly interact with the hydroquinone. Furthermore the side chains D278 and E81 were shown to be important interaction partners for hydroquinone oxidation. The residues of the QP-site are all highly involved in hydrogen bond network interactions

X-ray structure of the dimeric cytochrome bc1 complex from the soil bacterium Paracoccus denitrificans at 2.7-Å resolution

AbstractThe respiratory cytochrome bc1 complex is a fundamental enzyme in biological energy conversion. It couples electron transfer from ubiquinol to cytochrome c with generation of proton motive force which fuels ATP synthesis. The complex from the α-proteobacterium Paracoccus denitrificans, a model for the medically relevant mitochondrial complexes, lacked structural characterization. We show by LILBID mass spectrometry that truncation of the organism-specific, acidic N-terminus of cytochrome c1 changes the oligomerization state of the enzyme to a dimer. The fully functional complex was crystallized and the X-ray structure determined at 2.7-Å resolution. It has high structural homology to mitochondrial complexes and to the Rhodobacter sphaeroides complex especially for subunits cytochrome b and ISP. Species-specific binding of the inhibitor stigmatellin is noteworthy. Interestingly, cytochrome c1 shows structural differences to the mitochondrial and even between the two Rhodobacteraceae complexes. The structural diversity in the cytochrome c1 surface facing the ISP domain indicates low structural constraints on that surface for formation of a productive electron transfer complex. A similar position of the acidic N-terminal domains of cytochrome c1 and yeast subunit QCR6p is suggested in support of a similar function. A model of the electron transfer complex with membrane-anchored cytochrome c552, the natural substrate, shows that it can adopt the same orientation as the soluble substrate in the yeast complex. The full structural integrity of the P. denitrificans variant underpins previous mechanistic studies on intermonomer electron transfer and paves the way for using this model system to address open questions of structure/function relationships and inhibitor binding

The obligate respiratory supercomplex from Actinobacteria

International audienceActinobacteria are closely linked to human life as industrial producers of bioactive molecules and as human pathogens. Respiratory cytochrome bcc complex and cytochrome aa3 oxidase are key components of their aerobic energy metabolism. They form a supercomplex in the actinobacterial species Corynebacterium glutamicum. With comprehensive bioinformatics and phylogenetic analysis we show that genes for cyt bcc-aa3 supercomplex are characteristic for Actinobacteria (Actinobacteria and Acidimicrobiia, except the anaerobic orders Actinomycetales and Bifidobacteriales). An obligatory supercomplex is likely, due to the lack of genes encoding alternative electron transfer partners such as mono-heme cyt c. Instead, subunit QcrC of bcc complex, here classified as short di-heme cyt c, will provide the exclusive electron transfer link between the complexes as in C. glutamicum. Purified to high homogeneity, the C. glutamicum bcc-aa3 supercomplex contained all subunits and cofactors as analyzed by SDS-PAGE, BN-PAGE, absorption and EPR spectroscopy. Highly uniform supercomplex particles in electron microscopy analysis support a distinct structural composition. The supercomplex possesses a dimeric stoichiometry with a ratio of a-type, b-type and c-type hemes close to 1:1:1. Redox titrations revealed a low potential bcc complex (EmISP = + 160 mV, EmbL = − 291 mV, EmbH = − 163 mV, Emcc = + 100 mV) fined-tuned for oxidation of menaquinol and a mixed potential aa3 oxidase (EmCuA = + 150 mV, Ema/a3 = + 143/+317 mV) mediating between low and high redox potential to accomplish dioxygen reduction. The generated molecular model supports a stable assembled supercomplex with defined architecture which permits energetically efficient coupling of menaquinol oxidation and dioxygen reduction in one supramolecular entity

Structuring the complexity of integrated landscape approaches into selectable, scalable, and measurable attributes

Integrated landscape approaches (ILA) aim to reconcile multiple, often competing, interests across agriculture, nature conservation, and other land uses. Recognized ILA design principles provide guidance for implementation, yet application remains challenging, and a strong performance evidence-base is yet to be formed. Through a critical literature review and focus group discussions with practitioners, we identified considerable diversity of ILA in actors, temporal, and spatial scales, inter alia. This diversity hampers learning from and steering ILA because of the intractable nature of the concept. Therefore, we developed a tool—an ‘ILA mixing board’—to structure the complexity of ILA into selectable and scalable attributes in a replicable way to allow planning, diagnosing, and comparing ILA. The ILA mixing board tool presents seven qualifiers, each representing a key attribute of ILA design and performance (for example, project flexibility, inclusiveness of the dialogue, and the centrality of the power distribution). Each qualifier has five (non-normative) outcome indicators that can be registered as present or absent. This process in turn guides planners, evaluators and other participating stakeholders involved in landscape management to diagnose the ILA type, or its performance. We apply the ILA mixing board to three ILA cases in Nicaragua, Madagascar, and the Congo Basin to show some of the many possible configurations of qualifiers on the mixing board. Further application of the tool would allow comparative analysis of the complexity of ILA in a structured and manageable way thereby enhancing the understanding of ILA performance and informing the development of evidence-based land use policy

Strategy games to improve environmental policymaking

While the scientific community documents environmental degradation and develops scenarios to identify the operational margins of system Earth, less attention is given to how decisions are made that steer the system in one direction or the other. We propose to use strategy games for this purpose, increasing the representation of human agency in scenario development and creating spaces for deliberation between different worldviews. Played by the right people, strategy games could help break free from established norms and support more transparent democratic dialogues, responding to the human and social limitations of current decision-making

Strategy games to improve environmental policymaking

While the scientific community documents environmental degradation and develops scenarios to identify the operational margins of system Earth, less attention is given to how decisions are made that steer the system in one direction or the other. We propose to use strategy games for this purpose, increasing the representation of human agency in scenario development and creating spaces for deliberation between different worldviews. Played by the right people, strategy games could help break free from established norms and support more transparent democratic dialogues, responding to the human and social limitations of current decision-making. The question is, who gets to play