The NDH-1-complex of Synechocystis PCC6803:Modular assembly and physiological relevance.

Die unter dem Begriff NDH-1-Komplex zusammengefassten NAD(P)H:Chinon-Oxidoreduktasen von Synechocystis PCC 6803 entsprechen funktionell und im Aufbau dem Respirations-Komplex I aus Bakterien (NUO) und sind verwandt mit dem Komplex I in Mitochondrien und dem NDH-1-Komplex aus Plastiden. Der bakterielle wie auch der mitochondriale Komplex I sind Bestandteil der Atmungskette. Der plastidäre NDH-1 ist sowohl am zyklischen Elektronentransport der Photosynthese als auch an der Chlororespiration beteiligt. Die cyanobakteriellen NDH-1-Komplexe nehmen eine Sonderstellung ein, da sie neben dem respiratorischen und dem photosynthetischen Elektronentransport auch noch an der CO2-Fixierung teilnehmen. Diese unterschiedlichen Funktionen werden durch einen modularen Aufbau ermöglicht, der zu unterschiedlichen NDH-1-Komplexen führt (S, M, L und MS). Ob es sich dabei unter anderem um Zwischenprodukte oder Zerfallsprodukte dieses labilen Komplexes handelt, ist noch offen. Die Erweiterung des NDH-1M zum NDH-1L-Komplex geschieht durch die Anlagerung der Untereinheiten NdhD und NdhF an dem membranständigen Teil. Von den NdhD-und NdhF-Untereinheiten sind im Genom von Synechocystis und der meisten Cyanobakterien mehrere Homologe vorhanden, die in bestimmten paarweisen Kombinationen eingesetzt werden und so die funktionelle Vielfalt ermöglichen. Die Beteiligung der Untereinheiten NdhD3 und NdhD4 an der CO2-Fixierung wurde bereits beschrieben. Die Lokalisierung dieser unterschiedlichen NDH-1-Komplexe gehört aber neben der genauen Funktionsweise und den immer noch nicht identifizierten NAD(P)H-reduzierenden Untereinheiten zu den offenen Fragen der Forschung am NDH-1-Komplex. In der vorliegenden Arbeit wurden Untersuchungen zur Bedeutung der verschiedenen NdhD-Untereinheiten (mit Fokus auf NdhD1 und NdhD2) für die Lokalisierung der NDH-1-Komplexe in Thylakoid- oder Plasmamembran und der ausgeübten physiologischen Funktion durchgeführt. Lokalisierungsstudien mit isolierten Membranen und Immungold-Analysen zeigten, dass die NDH-1-Komplexe (M und L) überwiegend in der Thylakoidmembran, in Spuren aber auch als NDH-1M in der Plasmamembran vorhanden sind. Im Verlauf dieser Arbeit wurden noch erforderliche ndhD-Mutanten hergestellt und charakterisiert, um gezielt das NdhD1, das zusammen mit dem NdhF1 und dem NDH-1M den NDH-1L bildet, zu untersuchen. Von diesen besitzt die D1234-Mutante keine der vier NdhD-Untereinheiten mehr. Western-Blot-Analysen nach nativer Proteinauftrennung im BN-Gel zeigten, dass die D1234-Mutante die erste beschriebene Synechocystis-Mutante ist, die nur noch den NDH-1M-Komplex assembliert. Mit dieser Methode und den neuen selbst hergestellten Mutanten gelang es erstmals, die unterschiedlichen NDH-1L- und NDH-1MS-Komplexe im BN-Gel eindeutig zu identifizieren. Dass der NDH-1M-Komplex auch aktiv und (wenn auch stark eingeschränkt) an der Reduktion des PQ-Pools beteiligt ist, konnte u.a. durch Messung der Reaktionsgeschwindigkeiten vom PS I mit dem Dual-PAM belegt werden. Bei dieser Mutante fällt auch die Akzeptorseiten-Limitierung des PS I geringer aus als in einer ebenfalls untersuchten Mutante ohne NDH-1-Komplex, d.h. der NDH-1M kann im geringen Umfang NADP+ als Elektronenakzeptor des PS I bereitstellen. Wachstumskurven von verschiedenen NDH-1-Mutanten machten deutlich, dass für ein ungestörtes photoautotrophes als auch mixotrophes Wachstum der NDH-1M alleine nicht ausreichend ist. Erst wenn durch das NdhD1 der NDH-1L-Komplex gebildet werden kann, ist ein dem WT vergleichbares Wachstum möglich. Dies gilt mit leichten Einschränkungen auch für das NdhD2 (Bestandteil des NDH-1L'). Phylogenetische Analysen ergaben zudem, dass die Verwandtschaft der als NdhD5 und NdhD6 annotierten Proteine zu den übrigen NdhD-Untereinheiten (NdhD1-NdhD4) so gering ausfällt, dass diese beiden wohl keine redundante Funktion ausüben können. Somit können sie aus dem Kreis der potentiellen NdhD-Untereinheiten ausgeschlossen werden und wurden nicht weiter analysiert.In Synechocystis PCC 6803 many NAD(P)H:quinone oxidoreductases are grouped under the umbrella term NDH-1-complex. They correspond functionally and structurally to the complex I of bacteria and are related to the complex I of mitochondria and the NDH-1-complex of plastids. Both the bacterial and the mitochondrial complex I take part in the respiratory chain. The plastidal NDH-1 is involved in chlororespiration and additionally in the cyclic electron transport of photosynthesis. The cyanobacterial NDH-1-complexes are unique because they participate in both respiration and photosynthesis and moreover in the fixation of carbon dioxide. These different functions were enabled by a modular assembly which leads to different NDH-1-complexes (S, M, L and MS). Wether these are intermediates or caused by decay is still a matter of debate. The NDH-1M can be enlarged to NDH-1L by the addition of the peripheral subunits NdhD and NdhF to the membranous part. Synechocystis and most other cyanobacteria have several homologues of these subunits in their genome, which are combined in defined pairs permitting the functional diversity. The participation of NdhD3 and NdhD4 in fixation of carbon dioxide was previously characterized, but the precise localization, the operation modes of the different complexes and the still unknown NAD(P)H oxidizing subunits remain open questions. In this study the relevance of different NdhD-subunits (with focus on NdhD1 and NdhD2) for the localization of the NDH-1 (L and MS) in thylakoids and plasma membrane were analyzed and their physiological function was characterized. Studies of localization with isolated membranes and immunogold assays showed that NDH-1 (M and L) appear predominantly in the thylakoids while traces of NDH-1M were observed in the plasma membrane. In the course of this thesis, several required ndhD-mutants were constructed and characterized to study NdhD1 in particular. This subunit composes the NDH-1L together with the NdhF1 and NDH-1M. The new quadruple-mutant D1234 lacks all four NdhD-subunits. Western blot analyzes after blue-native electrophoresis of membranous proteins confirmed, that the D1234 is the first described mutant of Synechocystis which exhibits only NDH-1M. This method combined with the new self-produced mutants allowed to identify the complexes NDH-1L and NDH-1MS in the blue native gel for the first time. The still assembled NDH-1M was shown to be active in the reduction of the PQ-pool, although with poor reaction rates. This was chiefly established with calculations of the reaction rate of PS I, measured with the Dual-PAM. The acceptor limitation of PS I in this mutant was shown to be less than a mutant without the NDH-1-complex, caused by the weak provision of NADP+ by the NDH-1M as an electron acceptor of PS I. Growth curves of different NDH-1-mutants illustrate the insufficiency of NDH-1M for photoautotroph or mixotroph growth. Growth rates comparable to the wild type are only possible with NDH-1L, with involvement of NdhD1. This is also valid – with some reservations –for NDH-1L' (with participation of NdhD2). Phylogenetic analysis showed that the relationship between the NdhD5 and NdhD6 subunits with the other NdhD homologues (NdhD1-NdhD4) is weaker than the relationship within this group, so the potential for these two subunits to provide a redundant functionality of the other four seems to be marginal. Hence they could be excluded from the group of potentially NdhD subunits and were not investigated further

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

AbstractThe cytochrome b6f 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 b6f 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

Evidence for Electron Transfer from the Bidirectional Hydrogenase to the Photosynthetic Complex I (NDH-1) in the Cyanobacterium Synechocystis sp. PCC 6803

The cyanobacterial bidirectional [NiFe]-hydrogenase is a pentameric enzyme. Apart from the small and large hydrogenase subunits (HoxYH) it contains a diaphorase module (HoxEFU) that interacts with NAD(P)+ and ferredoxin. HoxEFU shows strong similarity to the outermost subunits (NuoEFG) of canonical respiratory complexes I. Photosynthetic complex I (NDH-1) lacks these three subunits. This led to the idea that HoxEFU might interact with NDH-1 instead. HoxEFUYH utilizes excited electrons from PSI for photohydrogen production and it catalyzes the reverse reaction and feeds electrons into the photosynthetic electron transport. We analyzed hydrogenase activity, photohydrogen evolution and hydrogen uptake, the respiration and photosynthetic electron transport of ΔhoxEFUYH, and a knock-out strain with dysfunctional NDH-1 (ΔndhD1/ΔndhD2) of the cyanobacterium Synechocystis sp. PCC 6803. Photohydrogen production was prolonged in ΔndhD1/ΔndhD2 due to diminished hydrogen uptake. Electrons from hydrogen oxidation must follow a different route into the photosynthetic electron transport in this mutant compared to wild type cells. Furthermore, respiration was reduced in ΔhoxEFUYH and the ΔndhD1/ΔndhD2 localization of the hydrogenase to the membrane was impaired. These data indicate that electron transfer from the hydrogenase to the NDH-1 complex is either direct, by the binding of the hydrogenase to the complex, or indirect, via an additional mediator

Evidence for Electron Transfer from the Bidirectional Hydrogenase to the Photosynthetic Complex I (NDH-1) in the Cyanobacterium Synechocystis sp. PCC 6803

The cyanobacterial bidirectional [NiFe]-hydrogenase is a pentameric enzyme. Apart from the small and large hydrogenase subunits (HoxYH) it contains a diaphorase module (HoxEFU) that interacts with NAD(P)+ and ferredoxin. HoxEFU shows strong similarity to the outermost subunits (NuoEFG) of canonical respiratory complexes I. Photosynthetic complex I (NDH-1) lacks these three subunits. This led to the idea that HoxEFU might interact with NDH-1 instead. HoxEFUYH utilizes excited electrons from PSI for photohydrogen production and it catalyzes the reverse reaction and feeds electrons into the photosynthetic electron transport. We analyzed hydrogenase activity, photohydrogen evolution and hydrogen uptake, the respiration and photosynthetic electron transport of ΔhoxEFUYH, and a knock-out strain with dysfunctional NDH-1 (ΔndhD1/ΔndhD2) of the cyanobacterium Synechocystis sp. PCC 6803. Photohydrogen production was prolonged in ΔndhD1/ΔndhD2 due to diminished hydrogen uptake. Electrons from hydrogen oxidation must follow a different route into the photosynthetic electron transport in this mutant compared to wild type cells. Furthermore, respiration was reduced in ΔhoxEFUYH and the ΔndhD1/ΔndhD2 localization of the hydrogenase to the membrane was impaired. These data indicate that electron transfer from the hydrogenase to the NDH-1 complex is either direct, by the binding of the hydrogenase to the complex, or indirect, via an additional mediator

Evidence for Electron Transfer from the Bidirectional Hydrogenase to the Photosynthetic Complex I (NDH-1) in the Cyanobacterium Synechocystis sp. PCC 6803

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