Conference Agenda

The nicotinamide nucleotide transhydrogenase catalyzes reversible hydride transfer between NADH and NADP⁺ and couples this reaction to the proton motive force to promote the generation of NADPH. This transhydrogenase is present in the mitochondrial inner membrane and in the cytoplasmic membranes of many bacteria including E. coli. The enzyme consists of three domains: domain I binds NAD(H); domain II contains multiple transmembrane helices and a proton channel; domain III binds NADP(H). Large conformational changes of the enzyme isolated from ovine mitochondria were previously observed in the presence of NADP⁺ or NADPH [1], but none of the conformations are compatible with hydride transfer from NADH to NADP⁺. In this work, the structure of the transhydrogenase from E. coli has been determined by cryo-electron microscopy, capturing multiple conformations in the presence of pairs of substrates/products: NAD⁺ & NADP⁺ and NADPH & NADP⁺. Most important is a conformation observed in the presence of a mixture of NADPH and NADP⁺ in which the binding sites for NADH (domain I) and NADP⁺ (domain III) are adjacent and compatible with direct hydride transfer. The structures, along with previous data, suggest a plausible mechanism for coupling the proton motive force to hydride transfer between NADH and NADP⁺.

[1] D. Kampjut, L.A. Sazanov, Structure and Mechanism of mitochondrial proton-translocating transhydrogenase, Nature, 573 (2019) 291-295.

Complex I is a gigantic redox-driven proton pump that catalyzes the NADH-driven reduction of quinones transducing the free energy into proton pumping across a biological membrane, >200 Å away from the active site [1,2]. Yet, despite major advances, its energy transduction mechanism remains poorly understood and much debated. Here we integrate biophysical, computational, and structural experiments to derive a unified molecular understanding of the long-range proton pumping mechanism in the Complex I machinery [3-8]. We show how the quinone chemistry triggers conformational and hydration changes in the membrane domain of Complex I activating the proton pump. Moreover, we dissect and engineer the minimal proton transport elements of the pumping machinery, and show how the proton translocation activity is achieved by electric field effects. Finally, we discuss how mutations, lipid interactions, and supercomplex formation modulate the activity and functionality of the machinery. On a general level, our findings show how bioenergetic enzymes employ similar fundamental principles to drive redox-driven ion transport and long-range coupling effects.

[1] Kaila, V. R. I.; Wikström, M. Architecture of bacterial respiratory chains. Nat. Rev. Microbiol. 19, (2021), 319−330.
[2] Kaila V. R. I. Accounts of Chemical Research 54, (2021), 4462-4473
[3] Beghiah A. et al. Kaila, V.R.I (2024, in review).
[4] Kaila, V.R.I (in preparation)
[5] Saura et al. Kaila, V.R.I (in preparation).
[6] Kim, H. et al. Kaila, V.R.I. Quinone Catalysis Modulates Proton Transfer Reactions in the Membrane Domain of Respiratory Complex I. J. Am. Chem. Soc. 145 (2023) 17075-17086.
[7] Pöverlein, M. et al. Kaila V.R.I. (in preparation)
[8] Sirohiwal, A; Gamiz-Hernandez, A. P.; Kaila, V. R. I. Mechanistic principles of hydrogen evolution in the membrane-bound hydrogenase, bioRxiv, 03.16.585322 (2024).

The most common primary mitochondrial diseases result from mutations of complex I. Inheritance of mutant NDUFS4, thought to support complex I assembly, causes partial complex I deficiency and the severe neuromuscular disease Leigh syndrome. However, even with complete deletion of NDUFS4 residual complex I activity remains in mice. We have investigated the state of assembly of complex I and respiratory supercomplexes isolated from livers of NDUFS4 mutant mice. We confirm holo-complex I only exists in supercomplexes, as do incompletely assembled complex I subassemblies. We determine the structure of supercomplexes containing complex I subassemblies lacking: 1) the N-module (N-less); and 2) entire peripheral arm (membrane domain alone). Thus, we observe stalled complex I assembly intermediates bound to complex III₂ and complex IV. These findings support the view that supercomplexes are early scaffolds of complex I assembly and not a late stage higher-order product forming after all complexes are fully assembled and thus, enhancing supercomplex formation may help mitigate complex I deficiency.

Respirasomes [1, 2] are self-sufficient molecular machines capable of not only transferring electrons from a donor to O₂, but also achieving energy conservation. Although metazoan respirasomes have been biochemically and structurally investigated [3], it is believed that bacterial counterparts do not exist. Here, I will present surprising new details regarding how my laboratory discovered the widespread occurrence of prokaryotic respirasomes. Notably, I will present convincing results spanning chemical microbiology, structural biochemistry, and evolutionary analysis to reveal the extraordinary mechanistic diversity that drives microbial aerobic respiration. Collectively, my talk will show how previously unknown bioenergetic strategies contribute to bacterial infallibility [4].

[1] Y. Hatefi, A.G. Haavik, D.E. Griffiths, Studies on the electron transfer system. XL. Preparation and properties of mitochondrial DPNH-coenzyme Q reductase. J Biol Chem 1962, 237 (1962) 1676-1680.

[2] H. Schägger, K. Pfeiffer, Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J 19 (2000) 1777-1783.

[3] J.A. Letts, K. Fiedorczuk, L.A. Sazanov, The architecture of respiratory supercomplexes. Nature 537 (2016) 644-648

[4] E.F. Gale, The Chemical Activities of Bacteria (3^rd ed), Academic Press, N.Y., 1951.