Complex I is a highly intricate membrane-bound protein complex that powers the cellular energy metabolism by a long-range ( > 300 Å) proton-coupled electron transfer (PCET) reaction. Here, we investigate the highly debated coupling mechanism of Complex I by probing the charge transfer reaction along its functionally central carboxylate pathway (E-channel). By combining biophysical and site-directed mutagenesis experiments with high-resolution (2.6-2.8 Å) cryo-electron microscopy (cryo-EM) and multiscale simulations, we identify a conserved carboxylate switch point (D79^NuoA) that mediates proton transfer by establishing a kinetic gate and couples the redox chemistry to proton pumping. We find that mutation of the identified site, as found in patients suffering from severe neurodegenerative disorders, drastically perturbs the charge transfer mechanism, and results in a 20% PCET activity. Our combined findings illustrate mechanistic principles of molecular gates underlying long-range charge transfer reactions, and show how disease mutations perturb the function of conserved switch points in energy transduction.

Site-direct mutagenesis provides a basis for functional studies of proteins. Yet, despite many available techniques, mutagenesis of large protein complexes can be tedious and highly challenging, with the replication fidelity and long-range amplification limiting the process. Here, we develop a method for site-directed mutagenesis of large protein complexes based on sub-fragmentation of its coding sequence that generates libraries of shorter sequences, where the DNA polymerase can accurately catalyse long-range elongation. Using the nuo genes encoding for the 0.5 MDa E. coli Complex I, we successfully dissect the 15.1 kb long DNA sequence into fragments of 900 bp with partially overlapping regions and unique primer sequences, creating a plasmid library that can be amplified by regular DNA polymerases. We showcase the method by sub-cloning the E. coli Complex I sequence into 20 fragments from a large pBAD expression vector (21.3 kb). The method shows a high success rate of introduced mutations, and an efficient Gibson assembly of the mutated fragments into the expression vector, enabling the accurate introduction of point-mutations into the large pBAD vector. We suggest that the plasmid sub-fragmentation provides an efficient method for mutagenesis of multi-subunit protein complexes with several homologous subunits, such as the respiratory Complex I, advancing mechanistic studies of large bioenergetics protein complexes.

Respiratory Complex I powers oxidative phosphorylation by a long-range proton-coupled electron transfer (PCET) reaction, with mutations linked to more than half of all human mitochondrial disorders. Yet, the molecular principles underlying the functional impairment remain difficult to test, as most mutations impede both the proton pumping and oxidoreductase activities due to the tightly coupled PCET process. Here, we probe how key disease mutations in the terminal ND5 subunit (NuoL/Nqo12), linked to the development of Leigh's syndrome (LS) and LHON/MELAS (F124L, M252T, D393N), affect the proton transport activity within the dissected antiporter module Nqo12. All constructs result in fully folded antiporter modules, with the introduced substitutions showing enhanced proton conduction rates across the proteoliposome membranes relative to the wild type module. Our molecular dynamics simulations reveal that the mutations perturb the internal water network and ion-pair dynamics that are central for the long-range PCET activity in Complex I. Taken together, we suggest that the mitochondrial disease mutations alter the redox-driven proton pumping activity of Complex I by perturbing the function of local proton gates, and result in an uncontrolled proton translocation across the antiporter module. The molecular consequences of disease mutations are discussed in the context of the proposed pumping mechanism.

Phosphorylation is among the most ubiquitous and essential posttranslational modifications in biological systems. It is regulated by highly complex enzymatic networks. Here, we explore enzyme-free phosphorylation in a designed peptide system. Specifically, we use phosphorylation to modulate coiled-coil (CC) assembly and dynamics. This exploits a nonbiological reaction cycle to phosphorylate and dephosphorylate a histidine residue in an α-helix, enabling or disabling CC formation with a second helix. Dephosphorylation is kinetically gated─it is 25× faster in the CC compared to the nonassembled state. As a result, the cycle of phosphorylation, CC formation, dephosphorylation, and CC disassembly is ratcheted. The minimal synthetic phosphorylation cycle provides design principles and fulfills requirements to perform work mimicking the function of molecular motors, walkers, and pumps.

Ribonucleotide reductases (RNRs) catalyze the conversion of ribonucleotide (RNA) to deoxyribonucleotide (DNA) building blocks initiated by a long-range (>30 Å) proton-coupled electron transfer (PCET) by mechanistic principles that remain much debated. By combining multiscale quantum and classical simulations with directed mutagenesis, X-ray crystallography, and vibrational and electron paramagnetic resonance spectroscopy, we elucidate here the molecular principles underlying how metal-free RNRs initiate the long-range PCET process by creating a highly stable 3,4-dihydroxyphenylalanine (DOPA) initiator radical. We show that DOPA• is redox-tuned by a low-barrier hydrogen bond (LBHB), with a delocalized proton that provides the catalytic power for the ribonucleotide reduction. We find that the LBHB couples to an extended hydrogen-bonded network, with distant mutations resulting in the loss of radical formation, and providing key molecular insight into the long-range radical transport mechanism in RNRs. On a general level, our findings support the direct involvement of LBHB in protein chemistry and the importance of quantum effects in enzyme catalysis.

The respiratory Complex I is a highly intricate redox-driven proton pump that powers oxidative phosphorylation across all domains of life. Yet, despite major efforts, its long-range energy transduction principles remain much debated. Here, we study the molecular principles of proton transport by engineering the antiporter modules of Complex I. By combining directed mutagenesis with time-resolved spectroscopy and molecular dynamics (MD) simulations, we identify conserved residues along the proton channels that control the rate of proton transfer across proteoliposome membranes. The antiporter modules catalyze this tightly regulated proton transport by transient water wires that follow intrinsic electric fields along the proton channels. Based on MD simulations, we identify conserved gating sites, established by nonpolar residues, which modulate the hydration and electric field effects underlying the proton transport upon mutation. On a general level, our findings highlight how the modular energy-transduction machinery of Complex I employs a combination of electrostatic and conformational coupling principles to catalyze long-range proton transport, with distinct similarities to other enzymes.

Mitochondrial membranes harbor the electron transport chain (ETC) that powers oxidative phosphorylation (OXPHOS) and drives the synthesis of ATP. Yet, under physiological conditions, the OXPHOS proteins operate as higher-order supercomplex (SC) assemblies, although their functional role remains poorly understood and much debated. By combining large-scale atomistic and coarse-grained molecular simulations with analysis of cryo-electron microscopic data and statistical as well as kinetic models, we show here that the formation of the mammalian I/III₂ supercomplex reduces the molecular strain of inner mitochondrial membranes by altering the local membrane thickness and leading to an accumulation of both cardiolipin and quinone around specific regions of the SC. We find that the SC assembly also affects the global motion of the individual ETC proteins with possible functional consequences. On a general level, our findings suggest that molecular crowding and strain effects provide a thermodynamic driving force for the SC formation, with a possible flux enhancement in crowded biological membranes under constrained respiratory conditions.

The bioenergetic complexes of energy-transducing membranes generate a proton current that powers the synthesis of adenosine triphosphate. Yet, since the early days of the chemiosmotic theory, it has remained elusive and much debated whether the proton motive force (PMF) delocalizes into the bulk solvent surrounding the energy-transducing membrane or if the thermodynamic force is exerted as a localized proton current along the membrane surface. To elucidate the molecular principles underlying protonation dynamics at biological membranes, we combine here proteoliposome experiments with fluorescence correlation spectroscopy and multiscale molecular simulations. We show that ubiquinone (Q10), which is an essential electron carrier of inner mitochondrial membranes, interacts with protons at the membrane, and alters the rate of the protonation reactions along the surface. We find that physiological Q10 concentrations increase the integrity of the liposome membranes to sustain a PMF and enhance the rate of surface protonation reactions of lipid-conjugated pH-sensitive fluorophores, occurring on a microsecond timescale. Our multiscale simulations reveal that the quinone headgroup localizes at the membrane surface and stabilizes protonated water species by cation–π and hydrogen-bonded interactions amplifying the proton exchange on the surface relative to the bulk solvent. We suggest that in addition to the well-established role of quinones as redox mediators in energy-transducing membranes, Q10 also promotes the proton-collecting antenna effect, mediating proton exchange along the membrane and supporting a local proton circuit model. Our combined findings provide molecular insight into propagation of proton currents along biological membranes and reveal key principles underlying the energy conversion mechanisms in biology.

Cytochrome bd is a terminal oxidase expressed under low oxygen conditions and central for the survival of many pathogens. Here, we characterize the cyt bd-II from Mycobacterium smegmatis, a member of a hitherto uncharacterized evolutionary group (qOR-2) of bd oxidases, by combining biochemical studies with cryo-electron microscopy (cryo-EM), and multiscale simulations. Overexpressing the appCB operon in its native host led to production of a highly active bd-II (k_obs = 30 e⁻ s⁻¹) that together with a high-resolution (2.8 Å) cryo-EM structure and multiscale simulations reveal unique proton pathways and oxygen channels responsible for its function. We propose that a pH-dependent molecular switch, involving coordination changes of heme d and surrounding bulky residues regulate substrate access into the active site. Taken together, our findings provide detailed mechanistic insight of qOR-2 type bd oxidases, and a basis for understanding the evolution of the superfamily.

Respiratory complex I is a central enzyme of cellular energy metabolism that couples electron transfer with proton translocation across a biological membrane. In doing so, it powers oxidative phosphorylation that drives energy consuming processes. Mutations in complex I lead to severe neurodegenerative diseases in humans. However, the biochemical consequences of these mutations remain largely unknown. Here, we use the Escherichia coli complex I as a model to biochemically characterize the F124LMT-ND5 mutation found in patients suffering from Leigh syndrome. We show that the mutation drastically perturbs proton translocation and electron transfer activities to the same extent, despite the remarkable 140 Å distance between the mutated position and the electron transfer domain. Our molecular dynamics simulations suggest that the disease-causing mutation induces conformational changes that hamper the propagation of an electric wave through an ion-paired network essential for proton translocation. Our findings imply that malfunction of the proton translocation domain is entirely transmitted to the electron transfer domain underlining the action-at-a-distance coupling in the proton-coupled electron transfer of respiratory complex I.