NADH:quinone oxidoreductase: the black box of the respiratory chain

Sammanfattning: Complex I or NADH:quinone oxidoreductase the largest, most complex and least understood of the five membrane-bound enzyme complexes constituting the mitochondrial respiratory chain. The enzyme is present in all types of organisms, from bacteria to mammals. The enzyme catalyses the oxidation of NADH produced by the citric acid cycle and reduction of lipid soluble quinone in the membrane. Flavin and a number of iron-sulfur clusters take part in the electron transfer through the enzyme, that is coupled to proton translocation across the membrane. The enzyme is also capable of catalysing the reverse reaction, i.e. DmH+ supported NAD+ reduction. No high resolution structure exists for this enzyme, and the mechanism of energy coupling is not understood. To learn more about the functional mechanism of Complex I and to be able to formulate an experimentally testable hypothesis for how the proton pumping machinery works, it is essential to know the number and location of the quinone binding sites. In this work we have investigated the action of Complex I specific inhibitors that interfere with quinone binding. Furthermore, we have synthesised azido-quinone analogues for direct photo-labelling of the quinone binding sites in Complex I. Three azido-quinone analogues were accepted as substrates by Complex I and exhibited normal inhibitor sensitivities, whereas only two of the compounds inactivated Complex I upon illumination. We have also used fusion protein techniques to determine the transmembrane topology of one protein subunit, that is a candidate for harbouring a quinone binding area. In mammalian mitochondria Complex I is composed of 46 different protein subunits whereas the bacterial enzyme consists of only 14 proteins. The simpler bacterial Complex I is an attractive model system, since biochemical and biophysical methods can be applied in combination with molecular biological techniques. But how generally applicable are the results? In this work we have made a detailed, in parallel comparison of the catalytic activities and inhibitor sensitivities of mammalian and bacterial Complex I. The role and function of the 32 accessory subunits in mammalian Complex I is poorly understood. Some subunits probably have a functional role, but often the accessory subunits are thought of as merely structural or insulatory. We have demonstrated that most, if not all, of the accessory Complex I subunits were present in the ancestral eukaryote evolving into plants, animals and fungi. We have found homologues to 4 accessory Complex I subunits in bacteria, but notably only in the a-proteobacteria, the group from which the ancestor of mitochondria arose.