http://www.dspace.cam.ac.uk:80/handle/1810/221768 Fri, 24 May 2013 18:01:45 GMT 2013-05-24T18:01:45Z http://www.dspace.cam.ac.uk:80/handle/1810/228700 Title: Structural and functional studies of mitochondrial NADH:ubiquinone oxidoreductase (complex I) Authors: King, Martin Abstract: NADH:ubiquinone oxidoreductase (complex I) is the largest and most complicated enzyme in the mitochondrial electron transfer chain. It catalyses the oxidation of NADH and the reduction of ubiquinone, coupled to the translocation of protons across the mitochondrial inner membrane, maintaining the proton motive force used for ATP synthesis. Complex I is the least understood of the respiratory enzymes; although the mechanisms of NADH oxidation and intramolecular electron transfer are gradually becoming appreciated, the mechanisms of quinone binding and reduction and proton translocation remain unknown. Complex I dysfunction has been implicated in a wide range of pathologies including mitochondrial diseases such as Leigh’s disease, as well as neurodegenerative diseases such as Alzheimer’s and Parkinson’s. The work described in the first part of this thesis is aimed at elucidating the structure of either a subcomplex of mitochondrial complex I, or of the intact enzyme itself. A comprehensive investigation revealed that hydrophilic subcomplexes of complex I from bovine heart mitochondria are not suitable for use as models of the intact enzyme. Attempts to prepare intact complex I of sufficient quality for structural work were successful; however, results from a large set of crystallization trials were disappointing. The second part of this thesis describes three studies of the function and mechanism of complex I from bovine heart mitochondria. First, the flavin mononucleotide, the site of NADH oxidation, was identified as the site of the ‘inhibitor-insensitive’ NADH:ubiquinone oxidoreduction reaction. The formation of semiquinones initiates redox cycling reactions with oxygen, producing vast amounts of reactive oxygen species; further studies revealed that other oxidants, such as paraquat, also react at the flavin site and initiate redox cycling reactions. Second, kinetic studies showed that the reaction between NADH and positively charged oxidants such as HAR (hexaammineruthenium (III)) proceeds by an unusual ternary reaction mechanism at the flavin site of complex I. Finally, double electron-electron resonance spectroscopy was used to show unambiguously that iron sulphur cluster 4Fe[TY]1 gives rise to electron paramagnetic resonance signal N4; the data provide an alternating potential energy profile for electron transfer along the cluster chain between the flavin and the quinone-binding site. Tue, 16 Nov 2010 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/228700 2010-11-16T00:00:00Z http://www.dspace.cam.ac.uk:80/handle/1810/217870 Title: Structural and biochemical studies of the regulation and catalytic mechanism of ATP synthase Authors: Bowler, Matthew William Abstract: ATP synthase (F1Fo-ATPase) catalyses the production of ATP from ADP and orthophosphate by using the proton motive force established across a membrane by photosynthesis or oxidative phosphorylation. The ATP synthase of eukaryotic mitochondria is located in the inner membrane and is comprised of two domains. The globular F1 domain protrudes into the matrix and it contains the catalytic sites for ATP synthesis. The membrane bound Fo domain contains a proton channel. The two domains are connected by central and peripheral stalks. When F1 is removed from the complex, it can hydrolyse ATP but not synthesise it. It is composed of nine subunits with the stoichiometry α3β3γδε . The α and β subunits are arranged alternately round the symmetric γ subunit, which, with the δ and ε subunits, forms the central stalk. Catalysis occurs by a rotary mechanism where rotation in Fo, induced by the passage of protons, is transmitted to F1 via the central stalk. The rotation of the γ subunit induces conformational changes in the catalytic β subunits that lead to the synthesis of ATP. The three catalytic sites proceed through three major and well defined conformations sequentially, and no two sites are the same at any one time. The peripheral stalk counters the tendency of the α3β3 subcomplex to rotate with the γ subunit. The ATP synthase of mitochondria is regulated by an inhibitor protein, IF1, that prevents hydrolysis of ATP when the proton motive force collapses. Saccharomyces cerevisiae has two inhibitor proteins, YIF1 and STF1. The states of oligomerisation of their active and inactive forms have been investigated. In contrast to bovine IF1, which is active as a dimer, the yeast inhibitors are active as monomers around pH 7.0. Like the bovine protein, they form inactive oligomers at higher pH values. While many of features of the mechanism of catalysis of the ATP synthase are well understood, it is now clear that there are many sub-steps within the cycle. Some of them have been revealed by analogues of phosphoryl transfer. Bovine mitochondrial F1-ATPase inhibited with ADP and magnesium fluoride forms a transition state analogue complex. Its structure was solved to 2.5 Å resolution. The βTP and βDP catalytic sites both contain ADP and MgF3 -. The βE subunit binds ADP, despite being in an essentially open conformation. The structure represents a new sub-step in the catalytic cycle just before the release of the substrates of ATP hydrolysis. Sat, 01 Jan 2005 00:00:00 GMT http://www.dspace.cam.ac.uk:80/handle/1810/217870 2005-01-01T00:00:00Z