Structure of NuoEF variants for the analysis of NADH binding by respiratory complex I

Abstract: Leigh syndrome is a common mitochondrial disorder that leads to severe physical and mental developmental disorders as well as a shortened life expectancy. A large share of the mutations associated with Leigh syndrome are located in the genes encoding for respiratory complex I, the first enzyme of the mitochondrial respiratory chain. A widely used model for mitochondrial complex I is the homologous complex from the bacterium Escherichia coli, which represents a structurally minimal form of the mammalian complex. Both complexes share the same overall L-shaped structure, the same structure in individual subunits, and possess the same cofactors. The only difference is the absence of an iron-sulfur center in the mitochondrial complex I, which is important for the stability of the bacterial complex. In contrast to the human complex, which consists of 45 different subunits, the E. coli complex has only 13 subunits.
Both complexes have homologous substrate binding sites and catalyze the same reaction, which is inhibited by the same inhibitors. This suggests that the bacterial complex I can serve as a suitable model for investigating the NADH:ubiquinone oxidoreductase of the mitochondria. Proteins from hyperthermophilic bacteria are often easily crystallizable. Due to the homology between E. coli complex I and the one from the hyperthermophilic bacterium Aquifex aeolicus, previous studies relied on the homologous subunits from A. aeolicus for X-ray structural analysis. It was observed that the structure of the NADH binding site in complex I of A. aeolicus corresponds to the NuoE and NuoF subunits, which are homologous to NDUFV2 (NuoE) and NDUFV1 (NuoF) subunits in Homo sapiens.
The aim of this study was to investigate the structural changes of four variants of the NDUFV1 subunit of mitochondrial complex I associated with Leigh syndrome. The mutations found in patients in NDUFV1 are R88G, E246K, P252R, and E377K. The structural analysis was conducted using variants of the homologous NuoF subunit from A. aeolicus with the corresponding mutations R66G (NDUFV1: R88G), E222K (NDUFV1: E246K), P228R (NDUFV1: P252R), and E345K (NDUFV1: E377K).
Using an established method for the production and purification of NuoEF, the four variants were successfully generated and their identity confirmed through SDS-PAGE. While three of the variants crystallized successfully, attempts to crystallize the E345KF variant were unsuccessful despite numerous crystallization attempts under different conditions. To characterize the effects of the mutations on the mechanism, the crystals were soaked in various solutions, some with and without reducing agents and substrate or product. Additionally, the crystals were soaked with NADH under anoxic conditions, with an increased exposure time to characterize the NADH binding within the crystal over time. Through these experiments, the effects of the NuoF mutations on the structural properties of the variants were analyzed, providing insights into the mechanism.
The data collection was conducted at the European Synchrotron Radiation Facility in Grenoble, France, and resulted in high resolution structures of the NuoF variants. Five structures were obtained from the R66G and P228R variants, while three structures were obtained from the E222K variant.
The mutations investigated in this study, all of which were found in patients with Leigh syndrome, affect product displacement rather than substrate binding. While the mutations alter the orientation of some amino acids in their vicinity, the hydrogen bonding networks of the active site are maintained. Only minor changes in the active site were observed in all variants, which collectively, however allow for a better product binding. This could lead to a dramatic reduction in turnover rate. Ultimately, the results shed further light on the mechanism of NADH oxidation by complex I.

Angaben zum Objekt

• Standort: Deutsche Nationalbibliothek Frankfurt am Main
• Umfang: Online-Ressource
• Sprache: Englisch
• Anmerkungen: Universität Freiburg, Dissertation, 2023

Klassifikation und Themen

• Schlagwort: NADH ; Escherichia coli ; Leigh-Encephalomyelopathie ; NADH-Dehydrogenase ; Atmungskette ; Kristallographie

Beteiligte, Orts- und Zeitangaben

• Ereignis: Veröffentlichung
• (wo): Freiburg
• (wer): Universität
• (wann): 2023
• Urheber: Göppert-Asadollahpour, Sanaz
• Beteiligte Personen und Organisationen: Plattner, Dietmar ; Friedrich, Thorsten ; Einsle, Oliver ; Albert-Ludwigs-Universität Freiburg. Fakultät für Chemie und Pharmazie

Weitere Informationen

• DOI: 10.6094/UNIFR/240286
• URN: urn:nbn:de:bsz:25-freidok-2402868
• Rechteinformation: Open Access; Der Zugriff auf das Objekt ist unbeschränkt möglich.
• Letzte Aktualisierung: 25.03.2025, 13:44 MEZ