Work

A modular mechanism determines the proton-pumping stoichiometry of respiratory complex I

Year: 2026

Type: preprint

Abstract: Respiratory complex I, a crucial metabolic enzyme, uses the reducing potential of NADH to pump protons across energy-transducing membranes, but its mechanism of catalysis remains unknown. Here, guided by detailed structural information, we combine targeted mutagenesis and biophysical analyses to define the proton-pumping pathways in the three antiporter-like (proton-pumping) subunits of complex I from Paracoccus denitrificans and Escherichia coli . In Pd -CI, a four-proton pump, one of the three proton-uptake pathways is inactive, reconciling the four-proton/three-subunit mismatch. In Ec -CI two pathways are inactive – and Ec -CI pumps only two protons. We define the structural determinants for redox-coupled proton uptake, establish a mechanistic framework for proton pumping, and reveal how the proton-pumping stoichiometry has been adapted to deliver efficient energy conservation in diverse bioenergetic environments.

Source: bioRxiv (Cold Spring Harbor Laboratory)

DOI: 10.64898/2026.10.08.757549

Authors: Robert A. Waddell, Harry L. Messent, Marcelo Mesa Costa-Lima, John J. Wright, Jack H. Salisbury, Judy Hirst

Institutions: University of Cambridge, MRC Mitochondrial Biology Unit, Medical Research Council

Language: English

Topic: Enzyme Structure and Function

Subfield: Structural Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: green

Funders: University of Bern, Gates Cambridge Trust, Medical Research Council

Awards: G1257286180, G1521966493, Mitochondrial complex I: An intricate energy-converting machine, a cornerstone of mitochondrial metabolism, and a locus of mitochondrial dysfunction and disease