The energy-converting mechanism of a modular biomachine: Uniting structure and function to establish the engineering principles of respiratory complex I

Complex I (NADH:ubiquinone oxidoreductase), one of the largest membrane-bound enzymes in the mammalian cell, uses the reducing potential of NADH to drive protons across the inner mitochondrial membrane and power ATP synthesis. It is a keystone of metabolism, crucial for survival of human cells, and a primary site of mitochondrial dysfunctions and clinical disease. Complex I is also an efficient energy-converting biomachine, which shares conserved subunits, a molecular architecture, and modular design with prokaryotic type-4 hydrogenases, such as formate hydrogenlyase (FHL), that contribute to microbial H2 metabolism and geochemical cycles. Detailed cryo-electron microscopy (cryoEM) structures have defined the physical framework of the catalytic machinery in complex I, but there are wide knowledge gaps in central aspects of how it works, and no firm foundation of functional data to distinguish competing mechanistic proposals. ModularBioMachine will combine advanced strategies for structure, nanoassembly, function and genetics to transform knowledge of the reversible energy-transducing mechanism of complex I and FHL, exploiting their similarities and differences for enhanced insights and impact. It will exploit two powerful complex I platforms, complex I-containing proteoliposomes, which enable cryoEM under precisely defined conditions during energy-linked catalysis, and complex I from Paracoccus denitrificans, which combines cryoEM, genetics and advanced functional analyses in a single system – and extend both to study FHL. Focussing on three crucial knowledge gaps, the power stroke at the heart of energy conversion, the clockwork mechanism that defines the moving parts, and the proton wiring diagrams, ModularBioMachine will deliver a coherent, integrated understanding of the shared engineering principles and molecular mechanism of energy conversion in these evolutionary-linked biomachines at an intersection of the mitochondrial and microbial worlds.