Sub-nanoskopische Ladungsverschiebungen im ubichinon-reduzierenden Zentrum N des Cytochrom-bc1-Komplexes von Rhodobacter capsulatus
Zusammenfassung der Projektergebnisse
The mechanism of the cytochrome bc complexes was studied by combining experimental (differential kinetic spectrophotometry) and theoretical (molecular dynamic simulations, phylogenomic and structural analyses) approaches. The electron/proton coupling in the cytochrome bc1 complex was studied with membrane vesicles isolated from cells of phototrophic alpha proteobacterim Rhodobacter capsulatus. Due to the presence of photosynthetic enzymes, it was possible to trigger the reactions in the cytochrome bc1 complex by flashes of light. Upon titrating the cytochrome bc1 complex by pyraclostrobin, a specific inhibitor of the quinol-oxidizing site P, it was found that the inhibitor, when added at only 100 nM, retarded the oxidation of cytochrome b and specifically slowed the voltage generation and the re-reduction of cytochromes c. When added at higher concentrations, pyraclostrobin fully blocked the turnover of the cytochrome bc1 complex, as expected. Since a flash-induced fast transient reduction of cytochrome b were observed in the presence of 100 nM of pyraclastrobin, at least one of the centers P was still functioning as fast as in an untreated enzyme. There are only two centers P in the dimeric cytochrome bc1 complex; therefore, by exclusion, the observed phenomena reflected the operation of the cytochrome bc1 complexes that contained a bound pyraclostrobin molecule in one of their two centers P. Hence, binding of a pyraclostrobin molecule to the one center P of a dimeric cytochrome bc1 complex did not prevent the oxidation of ubiquinol in the other center P but retarded several subsequent reactions that were coupled with the oxidation of cytochrome b. This could be considered as an evidence that the oxidation of cytochrome via center N of the cytochrome bc1 complex can be controlled by the state of the center P. By performing steered molecular dynamics simulations of the cytochrome bc1 complex, the conformational modes that could be responsible for the coupling between centers P and N in the same monomer and between centers P of different monomers were identified. It was shown that the position of the FeS-cluster-carrying domain of the Rieske protein is sensitive to the occupation state of center P. The evolutionary history of the cytochrome bc complexes, from their early spread among prokaryotic lineages and up to the mitochondrial cytochrome bc1 complex (complex III) and its role in apoptosis, could be reconstructed. The results of phylogenomic analysis suggest that the bacterial cytochrome b6f-type complexes with short cytochromes b were the ancient form that preceded in evolution the cytochrome bc1-type complexes with long cytochromes b. Specifically, a detailed scenario of the gradual involvement of the cardiolipin-containing mitochondrial cytochrome bc1 complex into the intrinsic apoptotic pathway has been proposed, where the functioning of the complex as an apoptotic trigger is viewed as a way to accelerate the elimination of the cells with irreparably damaged, ROS-producing mitochondria. The same in silico approach was applied o to other energy-converting systems, specifically, to different ATPases and GTPases. Analysis of the membrane rotary ATPases provided evidence for the primacy of sodium-dependent membrane bioenergetic. Analysis of ribosomal GTPases contributed to the reconstruction of the habitats of the first cells. The respective article (A.Y. Mulkidjanian, A.Y. Bychkov, D.V. Dibrova, M.Y. Galperin, E.V. Koonin, PNAS 109 (2012) E821-830) was featured in numerous media all over the world.
Projektbezogene Publikationen (Auswahl)
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Inventing the dynamo machine: the evolution of the F-type and V-type ATPases, Nature Rev Microbiol, 5 (2007) 892-899
A.Y. Mulkidjanian, K.S. Makarova, M.Y. Galperin, E.V. Koonin
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Proton translocation by the cytochrome bc1 complexes of phototrophic bacteria: introducing the activated Q-cycle, Photochem Photobiol Sci, 6 (2007) 19-34
A.Y. Mulkidjanian
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Evolutionary primacy of sodium bioenergetics, Biol Direct, 3 (2008) 13
A.Y. Mulkidjanian, M.Y. Galperin, K.S. Makarova, Y.I. Wolf, E.V. Koonin
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The past and present of sodium energetics: may the sodium-motive force be with you, Biochim Biophys Acta, 1777 (2008) 985-992
A.Y. Mulkidjanian, P. Dibrov, M.Y. Galperin
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Co-evolution of primordial membranes and membrane proteins, Trends Biochem Sci, 34 (2009) 206-215
A.Y. Mulkidjanian, M.Y. Galperin, E.V. Koonin
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Activated Q-cycle as a common mechanism for cytochrome bc1 and cytochrome b6f complexes, Biochim Biophys Acta, 1797 (2010) 1858-1868
A.Y. Mulkidjanian
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Characterization of the N-ATPase, a distinct, laterally transferred Na+-translocating form of the bacterial F-type membrane ATPase, Bioinformatics, 26 (2010) 1473-1476
D.V. Dibrova, M.Y. Galperin, A.Y. Mulkidjanian
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Origin of first cells at terrestrial, anoxic geothermal fields, Proc Natl Acad Sci USA, 109 (2012) E821-830
A.Y. Mulkidjanian, A.Y. Bychkov, D.V. Dibrova, M.Y. Galperin, E.V. Koonin
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Evolution of cell division: from shear mechanics to complex molecular machineries, Cell, 152 (2013) 942-944
E.V. Koonin, A.Y. Mulkidjanian
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Evolution of cytochrome bc complexes: from membrane-anchored dehydrogenases of ancient bacteria to triggers of apoptosis in vertebrates, Biochim Biophys Acta, 1827 (2013) 1407-1427
D.V. Dibrova, D.A. Cherepanov, M.Y. Galperin, V.P. Skulachev, A.Y. Mulkidjanian
