Powerful proteins : structure and function of catalytic subunits of electrogenic NADH:quinone oxidoreductases

Electrogenic NADH:quinone oxidoreductases are large, membrane-embedded enzyme complexes found in the respiratory chain of prokaryotes and the mitochondria of eukaryotes. They represent the first module of the oxidative phosphorylation system which converts the energy from nutrients into an electrochemical gradient by coupling redox reactions to the translocation of cations across membranes. A long chain of events, such as the synthesis of ATP, ion homeostasis, reactive oxygen species production and bacterial motility depend on the activity of these complexes. Complex I consists of up to 45 subunits and can be found in the inner mitochondrial membrane of eukaryotes and in prokaryotes, where it is called NDH I. We investigated the isolated, hydrophobic ND5 subunit, which shows homologies to cation/proton antiporters, from human or Yarrowia lipolytica complex I. In vivo and biochemical analyses provided data on the cation translocation function and the alteration of function by disease-associated mutations. Taken together with the recently published 3D structure of bacterial complex I, these data allowed us to demonstrate that the ND5 subunit could possibly act as an antiporter module of mitochondrial complex I. Sodium ion translocating NADH:quinone oxidoreductase (Na+-NQR) is an enzyme found in many pathogenic bacteria. It consists of six subunits (NqrA - NqrF) whose 3D structures and enzymatic mechanisms were not known in detail at the time this project was initiated. By using high-resolution X-ray structures and site-directed mutagenesis, combined with biochemical studies, we proposed a model for catalysis and substrate selectivity on the atomic level of the electron input module of the complex, the NADH oxidizing domain of subunit NqrF. Furthermore, we analyzed the binding of silver ions to a cysteine residue in the NADH binding pocket and found that it leads to the inhibition of the Na+-NQR and to the killing of Vibrio cholerae in the nanomolar range. Subunit NqrA forms part of the quinone reductase module. By the use of physicochemical and biochemical methods we identified the herbicide 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB) as a quinone antagonist and inhibitor of the Na+-NQR complex and discovered two adjacent quinone binding sites on NqrA.Elektrogene NADH:Chinon Oxidoreduktasen sind grosse, in die Membran eingebettete Enzym-Komplexe der Atmungskette von Prokaryoten und Eukaryoten. Sie repräsentieren das erste Modul der oxidativen Phosphorylierung, welche die Energie aus Nährstoffen in einen elektrochemischen Gradienten wandelt, indem Redox-Reaktionen an den Transport von Kationen über eine Membran gekoppelt werden. Eine lange Kette an Abläufen in der Zelle hängt von der Aktivität dieser Komplexe ab, so z.B. die Synthese von ATP, Ionen-Homöostase, Produktion von reaktiven Sauerstoffspezies und Motilität von Bakterien. Komplex I besteht aus bis zu 45 Untereinheiten und findet sich in der inneren Mitochondrienmembran von Eukaryoten und in Prokaryoten, wo er als NDH I bekannt ist. Wir haben die isolierte, hydrophobe ND5 Untereinheit des Komplex I vom Menschen und der Hefe Yarrowia lipolytica untersucht, welche Homologien aufweist zu Kationen/Protonen Antiportern. In vivo und biochemische Analysen ermöglichten die funktionelle Untersuchung der Kationen-Translokation und der Veränderung der Funktion durch Krankheits-assoziierte Mutationen. Zusammen mit der kürzlich publizierten 3D Struktur des bakteriellen Komplex I haben es uns diese Daten erlaubt zu zeigen, dass die ND5 Untereinheit als Antiporter im mitochondriellen Komplex I fungieren könnte. Die Na+-translozierende NADH:Chinon Oxidoreduktase (Na+-NQR) wird häufig in pathogenen Bakterien vorgefunden. Sie besteht aus sechs Untereinheiten (NqrA - NqrF), deren 3D Struktur und Funktion zu Beginn dieses Projekts nicht im Detail bekannt waren. Durch hochauflösende Röntgen-Kristallstrukturen und ortsgerichtete Mutagenese sowie mittels biochemischen Methoden ist es uns gelungen, auf der atomaren Ebene ein Modell für die Katalyse und die Substratselektivität des Elektroneninput-Moduls, der NADH oxidierenden Domäne der Untereinheit NqrF, zu generieren. Wir haben weiterhin die Bindung von Silberionen an ein Cystein der NADH-Bindetasche analysiert und festgestellt, dass nanomolare Konzentrationen von Ag+ durch diese Bindung zur Hemmung der Na+-NQR und zum Abtöten von Vibrio cholerae führen. Die Untereinheit NqrA bildet einen Teil des Chinon- reduzierenden Moduls. Durch physikochemische und biochemische Untersuchungen konnten wir das Pflanzenschutzmittel 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinon (DBMIB) als Antagonisten zu Chinon und Inhibitor der Na+-NQR identifizieren, sowie zwei nebeneinanderliegende Chinon-Bindestellen auf NqrA charakterisieren

Exploratory factor and principal component analyses: some new aspects

Exploratory Factor Analysis (EFA) and Principal Component Analysis (PCA) are popular techniques for simplifying the presentation of, and investigating the structureof, an (n × p) data matrix. However, these fundamentally different techniques are frequently confused, and the differences between them are obscured, because they give similar results in some practical cases. We therefore investigate conditions under which they are expected to be close to each other, by considering EFA as a matrix decomposition so that it can be directly compared with the data matrix decomposition underlying PCA. Correspondingly, we propose an extended version of PCA, called the EFA-like PCA, which mimics the EFA matrix decomposition in the sense that they contain the same unknowns. We provide iterative algorithms for estimating the EFA-like PCA parameters, and derive conditions that have to be satisfied for the two techniques to give similar results. Throughout, we consider separately the cases n > p and p ≥ n. All derived algorithms and matrix conditions are illustrated on two data sets, one for each of these two cases

NMR Reveals Double Occupancy of Quinone-type Ligands in the Catalytic Quinone Binding Site of the Na⁺-translocating NADH : Quinone Oxidoreductase from Vibrio cholerae

The sodium ion-translocating NADH:quinone oxidoreductase (Na+-NQR) from the pathogen Vibrio cholerae exploits the free energy liberated during oxidation of NADH with ubiquinone to pump sodium ions across the cytoplasmic membrane. The Na+-NQR consists of four membrane-bound subunits NqrBCDE and the peripheral NqrF and NqrA subunits. NqrA binds ubiquinone-8 as well as quinones with shorter prenyl chains (ubiquinone-1 and ubiquinone-2). Here we show that the quinone derivative 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), a known inhibitor of the bc1 and b6f complexes found in mitochondria and chloroplasts, also inhibits quinone reduction by the Na+-NQR in a mixed inhibition mode. Tryptophan fluorescence quenching and saturation transfer difference NMR experiments in the presence of Na+-NQR inhibitor (DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide) indicate that two quinone analog ligands are bound simultaneously by the NqrA subunit with very similar interaction constants as observed with the holoenzyme complex. We conclude that the catalytic site of quinone reduction is located on NqrA. The two ligands bind to an extended binding pocket in direct vicinity to each other as demonstrated by interligand Overhauser effects between ubiquinone-1 and DBMIB or 2-n-heptyl-4-hydroxyquinoline N-oxide, respectively. We propose that a similar spatially close arrangement of the native quinone substrates is also operational in vivo, enhancing the catalytic efficiency during the final electron transfer steps in the Na+-NQR

Conformational coupling of redox-driven Na+ -translocation in Vibrio cholerae NADH:quinone oxidoreductase

In the respiratory chain, NADH oxidation is coupled to ion translocation across the membrane to build up an electrochemical gradient. In the human pathogen Vibrio cholerae, the sodium-pumping NADH:quinone oxidoreductase (Na+-NQR) generates a sodium gradient by a so far unknown mechanism. Here we show that ion pumping in Na+-NQR is driven by large conformational changes coupling electron transfer to ion translocation. We have determined a series of cryo-EM and X-ray structures of the Na+-NQR that represent snapshots of the catalytic cycle. The six subunits NqrA, B, C, D, E, and F of Na+-NQR harbor a unique set of cofactors that shuttle the electrons from NADH twice across the membrane to quinone. The redox state of a unique intramembranous [2Fe-2S] cluster orchestrates the movements of subunit NqrC, which acts as an electron transfer switch. We propose that this switching movement controls the release of Na+ from a binding site localized in subunit NqrB.ISSN:1545-9993ISSN:1545-998

Organelle-specific expression of subunit ND5 of human complex I (NADH dehydrogenase) alters cation homeostasis in Saccharomyces cerevisiae

The ND5 component of the respiratory complex I is a large, hydrophobic subunit encoded by the mitochondrial genome. Its bacterial homologue, the NDH-1 subunit NuoL, acts as a cation transporter in the absence of other NDH-1 subunits. Mutations in human ND5 are frequently observed in neurodegenerative diseases. Wild type and mutant variants of ND5 fused to GFP or a FLAG peptide were targeted to the endoplasmatic reticulum (ER) or the inner mitochondrial membrane of Saccharomyces cerevisiae, which lacks an endogenous complex I. The localization of ND5 fusion proteins was confirmed by microscopic analyses of S. cerevisiae cells, followed by cellular fractionation and immunostaining. The impact of the expression of ND5 fusion proteins on the growth of S. cerevisiae in the presence and absence of added salts was studied. ER-resident ND5 conferred Li(+) sensitivity to S. cerevisiae, which was lost when the E145V variant of ND5 was expressed. All variants of ND5 tested led to increased resistance of S. cerevisiae at high external concentrations of Na(+) or K(+). The data seem to indicate that ND5 influences the salt homeostasis of S. cerevisiae independent of other complex I subunits, and paves the way for functional studies of mutations found in mitochondrially encoded complex I genes