Open Access

Maja Aleksandra Tocilescu

• The NADH:ubiquinone oxidoreductase (complex I) is a large membrane bound protein complex coupling the redox reaction of NADH oxidation and quinone reduction to vectorial proton translocation across bioenergetic membranes. The mechanism of proton pumping is still unknown; it seems however that the reduction of quinone induces conformational changes which drive proton uptake from one side and release at the other side of the membrane. In this study the proposed quinone and inhibitor binding pocket located at the interface of the 49-kDa and PSST subunits was explored by a large number of point mutations introduced into complex I from the strictly aerobic yeast Yarrowia lipolytica. Point mutations were systematically chosen based on the crystal structure of the hydrophilic domain of complex I from Thermus thermophilus. In total, the properties of 94 mutants at 39 positions which completely cover the lining of the large putative quinone and inhibitor binding cavity are described and discussed here. A structure/function analysis allowed the identification of functional domains within the large putative quinone binding cavity. A possible quinone access path ranging from the N-terminal beta-sheet of the 49-kDa subunit into the pocket to tyrosine 144 could be defined, since all exchanges introduced here, caused an almost complete loss of complex I activity. A region located deeper in the proposed quinone binding pocket is apparently not important for complex I activity. In contrast, all exchanges of tyrosine 144, even the very conservative mutant Y144F, essentially abolished dNADH:DBQ oxidoreductase activity of complex I. However, with higher concentrations of Q1 or Q2 the dNADH:Q oxidoreductase activity was largely restored in the mutants with the more conservative exchanges. Proton pumping experiments showed that this activity was also coupled to proton translocation, indicating that these quinones were reduced at the physiological site. However, the apparent Km values for Q1 or Q2 were drastically increased, clearly demonstrating that tyrosine 144 is central for quinone binding and reduction. These results further prove that the enzymatically relevant quinone binding site of complex I is located at the interface of the 49-kDa and PSST subunits. The quinone binding pocket is thought to comprise the binding sites for a plethora of specific complex I inhibitors that are usually grouped into three classes. The large array of mutants targeting the quinone binding cavity was examined with a representative of each inhibitor class. Many mutants conferring resistance were identified which, depending on the inhibitor tested, clustered in well defined and partially overlapping regions of the large putative quinone and inhibitor binding cavity. Mutants with effects on type A (DQA) and type B (rotenone) inhibitors were found in a subdomain corresponding to the former [NiFe] site in homologous hydrogenases, whereby the type A inhibitor DQA seems to bind deeper in this domain. Mutants with effects on the type C inhibitor (C12E8) were found in a narrow crevice. Exchanging more exposed residues at the border of these well defined domains affected all three inhibitor types. Therefore, the results as a whole provide further support for the concept that different inhibitor classes bind to different but partially overlapping binding sites within a single large quinone binding pocket. In addition, they also indicate the approximate location of the binding sites within the structure of the large quinone and inhibitor binding cavity at the interface of the 49 kDa and the PSST subunit. It has been proposed earlier that the highly conserved HRGXE-motif in the 49-kDa subunit forms a part of the quinone binding site of complex I. Mutagenesis of the HRGXE-motif, revealed that these residues are rather critical for complex I assembly and seem to have an important structural role. The question why iron-sulfur cluster N1a is not detectable by EPR in many models organisms is not solved yet. Introducing polar and positively charged amino acid residues close to this cluster in order to increase its midpoint potential did not result in the appearance of the cluster N1a EPR signal in mitochondrial membranes from the mutants. Clearly, further research will be necessary to gain insights to the function of this iron-sulfur cluster in complex I. In an additional project, a new and simple in vivo screen for complex I deficiency in Y. lipolytica was developed and optimized. This assay probes for defects in complex I assembly and stability, oxidoreductase activity and also proton pumping activity by complex I. Most importantly, this assay is applicable to all Y. lipolytica strains and could be used to identify loss-of-function mutants, gain-of-functions mutants (i.e. resistance towards complex I inhibitors) and revertants due to mutations in both nuclear and mitochondrially encoded genes of complex I subunits.
• Komplex I (NADH:Ubichinon Oxidoreduktase) ist der größte und gleichzeitig auch der am wenigsten verstandene Teil der mitochondrialen Atmungskette. Der membran-ständige Enzymkomplex katalysiert eine Redoxreaktion, bei der Elektronen von NADH auf Ubichinon übertragen werden; wie diese Redoxreaktion an die Proton¬entrans-lokation durch die innere Mitochondrienmembran gekoppelt ist, ist jedoch bisher völlig unbekannt. Neueste Ergebnisse deuten darauf hin, dass die Reduktion des Ubichinons Konformationsänderungen im Membranteil von Komplex I induziert, die schließlich die Protonentranslokation bewerkstelligen. In der vorliegenden Arbeit wurde die Ubichinonbindetasche systematisch mittels struktur-basierter Mutagenese charakterisiert. Als Modellorganismus diente dabei die obligat aerobe Hefe Yarrowia lipolytica. Die Ergebnisse erlaubten die Unterteilung dieser weitläufigen Chinonbindetasche in funktionelle Domänen. So wurde ein Bereich identifiziert, der offenbar als Zugang für das Chinonsubstrat fungiert, während das Tyrosin 144 an der Bindung und Reduktion des Chinons beteiligt ist. Andere Bereiche tiefer in der Bindetasche sind hingegen für die Chinonreduktion gänzlich irrelevant. Mit Hilfe von Komplex I spezifischen Inhibitoren konnte gezeigt werden, dass die drei Hauptklassen von Komplex I Inhibitoren tatsächlich alle an teilweise überlappende Bindestellen innerhalb der großen Chinonbindetasche binden. Es war vermutet worden, dass das streng konservierte HRGXE-Motiv an der Chinonbindung beteiligt ist; jedoch deuten die Mutageneseergebnisse dieser Arbeit darauf hin, dass dieses Motiv vor allem eine strukturelle Funktion erfüllt. Die Funktion des Eisen-Schwefel-Zentrums N1a der 24-kDa Untereinheit ist ungeklärt, doch wird spekuliert, dass es die Produktion von reaktiven Sauerstoffspezies durch Komplex I reguliert. Durch das Einfügen von polaren und positiv geladenen Aminosäureresten in die Nähe des Eisen-Schwefel-Zentrums sollte das Mittelpunkts-potential angehoben werden. Die Aus¬wirkungen der Mutationen sollen in Kooperation mit der Arbeitsgruppe von Dr. Judy Hirst (MRC, Dunn Human Nutrition Unit, Cambridge, England) analysiert werden. In einem separaten Teilprojekt wurde ein neuer und einfacher in vivo Screen zur Identifizierung von Komplex I defizienten Y. lipolytica Stämmen entwickelt. Dieser Screen erlaubt die Untersuchung aller Y. lipolytica Mutanten auf Komplex I Gehalt, Redoxaktivität sowie Protonentranslokationsaktivität.