@phdthesis{12781,
  abstract     = {Most energy in humans is produced in form of ATP by the mitochondrial respiratory chain consisting of several protein assemblies embedded into lipid membrane (complexes I-V). Complex I is the first and the largest enzyme of the respiratory chain which is essential for energy production. It couples the transfer of two electrons from NADH to ubiquinone with proton translocation across bacterial or inner mitochondrial membrane. The coupling mechanism between electron transfer and proton translocation is one of the biggest enigma in bioenergetics and structural biology. Even though the enzyme has been studied for decades, only recent technological advances in cryo-EM allowed its extensive structural investigation. 

Complex I from E.coli appears to be of special importance because it is a perfect model system with a rich mutant library, however the structure of the entire complex was unknown. In this thesis I have resolved structures of the minimal complex I version from E. coli in different states including reduced, inhibited, under reaction turnover and several others. Extensive structural analyses of these structures and comparison to structures from other species allowed to derive general features of conformational dynamics and propose a universal coupling mechanism. The mechanism is straightforward, robust and consistent with decades of experimental data available for complex I from different species. 

Cyanobacterial NDH (cyanobacterial complex I) is a part of broad complex I superfamily and was studied as well in this thesis. It plays an important role in cyclic electron transfer (CET), during which electrons are cycled within PSI through ferredoxin and plastoquinone to generate proton gradient without NADPH production. Here, I solved structure of NDH and revealed additional state, which was not observed before. The novel “resting” state allowed to propose the mechanism of CET regulation. Moreover, conformational dynamics of NDH resembles one in complex I which suggest more broad universality of the proposed coupling mechanism.

In summary, results presented here helped to interpret decades of experimental data for complex I and contributed to fundamental mechanistic understanding of protein function.
},
  author       = {Kravchuk, Vladyslav},
  isbn         = {978-3-99078-029-9},
  issn         = {2663-337X},
  pages        = {127},
  publisher    = {Institute of Science and Technology Austria},
  title        = {{Structural and mechanistic study of bacterial complex I and its cyanobacterial ortholog}},
  doi          = {10.15479/at:ista:12781},
  year         = {2023},
}

@phdthesis{8340,
  abstract     = {Mitochondria are sites of oxidative phosphorylation in eukaryotic cells. Oxidative phosphorylation operates by a chemiosmotic mechanism made possible by redox-driven proton pumping machines which establish a proton motive force across the inner mitochondrial membrane. This electrochemical proton gradient is used to drive ATP synthesis, which powers the majority of cellular processes such as protein synthesis, locomotion and signalling. In this thesis I investigate the structures and molecular mechanisms of two inner mitochondrial proton pumping enzymes, respiratory complex I and transhydrogenase. I present the first high-resolution structure of the full transhydrogenase from any species, and a significantly improved structure of complex I. Improving the resolution from 3.3 Å available previously to up to 2.3 Å in this thesis allowed us to model bound water molecules, crucial in the proton pumping mechanism. For both enzymes, up to five cryo-EM datasets with different substrates and inhibitors bound were solved to delineate the catalytic cycle and understand the proton pumping mechanism. In transhydrogenase, the proton channel is gated by reversible detachment of the NADP(H)-binding domain which opens the proton channel to the opposite sites of the membrane. In complex I, the proton channels are gated by reversible protonation of key glutamate and lysine residues and breaking of the water wire connecting the proton pumps with the quinone reduction site. The tight coupling between the redox and the proton pumping reactions in transhydrogenase is achieved by controlling the NADP(H) exchange which can only happen when the NADP(H)-binding domain interacts with the membrane domain. In complex I, coupling is achieved by cycling of the whole complex between the closed state, in which quinone can get reduced, and the open state, in which NADH can induce quinol ejection from the binding pocket. On the basis of these results I propose detailed mechanisms for catalytic cycles of transhydrogenase and complex I that are consistent with a large amount of previous work. In both enzymes, conformational and electrostatic mechanisms contribute to the overall catalytic process. Results presented here could be used for better understanding of the human pathologies arising from deficiencies of complex I or transhydrogenase and could be used to develop novel therapies.},
  author       = {Kampjut, Domen},
  isbn         = {978-3-99078-008-4},
  issn         = {2663-337X},
  pages        = {242},
  publisher    = {Institute of Science and Technology Austria},
  title        = {{Molecular mechanisms of mitochondrial redox-coupled proton pumping enzymes}},
  doi          = {10.15479/AT:ISTA:8340},
  year         = {2020},
}

@phdthesis{8353,
  abstract     = {Mrp (Multi resistance and pH adaptation) are broadly distributed secondary active antiporters that catalyze the transport of monovalent ions such as sodium and potassium outside of the cell coupled to the inward translocation of protons. Mrp antiporters are unique in a way that they are composed of seven subunits (MrpABCDEFG) encoded in a single operon, whereas other antiporters catalyzing the same reaction are mostly encoded by a single gene. Mrp exchangers are crucial for intracellular pH homeostasis and Na+ efflux, essential mechanisms for H+ uptake under alkaline environments and for reduction of the intracellular concentration of toxic cations. Mrp displays no homology to any other monovalent Na+(K+)/H+ antiporters but Mrp subunits have primary sequence similarity to essential redox-driven proton pumps, such as respiratory complex I and membrane-bound hydrogenases. This similarity reinforces the hypothesis that these present day redox-driven proton pumps are descended from the Mrp antiporter. The Mrp structure serves as a model to understand the yet obscure coupling mechanism between ion or electron transfer and proton translocation in this large group of proteins. In the thesis, I am presenting the purification, biochemical analysis, cryo-EM analysis and molecular structure of the Mrp complex from Anoxybacillus flavithermus solved by cryo-EM at 3.0 Å resolution. Numerous conditions were screened to purify Mrp to high homogeneity and to obtain an appropriate distribution of single particles on cryo-EM grids covered with a continuous layer of ultrathin carbon. A preferred particle orientation problem was solved by performing a tilted data collection. The activity assays showed the specific pH-dependent
profile of secondary active antiporters. The molecular structure shows that Mrp is a dimer of seven-subunit protomers with 50 trans-membrane helices each. The dimer interface is built by many short and tilted transmembrane helices, probably causing a thinning of the bacterial membrane. The surface charge distribution shows an extraordinary asymmetry within each monomer, revealing presumable proton and sodium translocation pathways. The two largest
and homologous Mrp subunits MrpA and MrpD probably translocate one proton each into the cell. The sodium ion is likely being translocated in the opposite direction within the small subunits along a ladder of charged and conserved residues. Based on the structure, we propose a mechanism were the antiport activity is accomplished via electrostatic interactions between the charged cations and key charged residues. The flexible key TM helices coordinate these
electrostatic interactions, while the membrane thinning between the monomers enables the translocation of sodium across the charged membrane. The entire family of redox-driven proton pumps is likely to perform their mechanism in a likewise manner.},
  author       = {Steiner, Julia},
  issn         = {2663-337X},
  pages        = {191},
  publisher    = {Institute of Science and Technology Austria},
  title        = {{Biochemical and structural investigation of the Mrp antiporter, an ancestor of complex I}},
  doi          = {10.15479/AT:ISTA:8353},
  year         = {2020},
}