The underlying molecular mechanisms of cooperativity and allosteric regulation are well understood for many proteins with hemoglobin and aspartate transcarbamoylase serving as prototypical examples 1,2 . Binding of effectors typically causes a structural transition of the protein that is propagated through signaling pathways to remote sites involving marked changes on the tertiary and sometimes even quaternary level [1][2][3][4][5] . However, the origin of these signals and the molecular mechanism of long-range signaling at an atomic level are a field of great dispute [5][6][7][8] . The different spatial and time scales involved in signaling pathways make the experimental observation challenging, in particular the positions and movement of mobile protons are invisible to current structural methods. Here, we report the first experimental observation of fluctuating low-barrier hydrogen bonds (LBHBs) as switching elements in cooperativity pathways of multimeric enzymes. We have observed these LBHBs in ultra-high resolution ( 1 Å) X-ray crystallographic structures of two multimeric enzymes, with their assignment validated through computational calculations. Catalytic events at the active sites switch between LBHBs and ordinary hydrogen bonds in a circuit consisting of acidic side chains and water molecules, transmitting a signal through the collective repositioning of protons much in the way of an atomistic Newtons cradle. The resulting communication synchronizes catalysis in the oligomer. Our studies provide multiple lines of evidence and a working model for not only the existence of LBHBs in proteins, but also for the first connection to enzyme cooperativity. This finding opens the door for whole new principles of drug and enzyme design, where one purposefully includes sequences of residues, which enable long-range communication and thus regulation of engineered biomolecules.The structural transitions associated with signaling in proteins are mediated by dedicated cooperativity and allosteric pathways, and commonly include the rupture and formation of salt bridges and hydrogen bonds [5][6][7][8] . In principle, signaling does not require large-scale motions and can operate via proton transfer through hydrogen bonds in conjugated proton wires as observed in e.g. water 9 . The latter involves the transient formation of LBHBs, whereby a proton is shared between two heteroatoms. LBHBs arise when the pKa values of two neighboring moieties are closely matched and the interatomic distance between the two heteroatoms are smaller than the sum of their van-der-Waals radii ( 2.55 Å for O-O pairs, 2.65 Å for O-N pairs) 10 . In enzyme function, LBHBs have been proposed to be part of charge relay networks at the active site, and to lower the activation barrier for substrate turnover 11,12 . However, ever since their initial proposal, the contribution of LBHBs in enzyme catalysis or even their mere existence has been fiercely debated. While some proponents have

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We recently reported the discovery of a lysine–cysteine redox switch in proteins with a covalent nitrogen–oxygen–sulfur (NOS) bridge. Here, a systematic survey of the whole protein structure database discloses that NOS bridges are ubiquitous redox switches in proteins of all domains of life and are found in diverse structural motifs and chemical variants. In several instances, lysines are observed in simultaneous linkage with two cysteines, forming a sulfur–oxygen–nitrogen–oxygen–sulfur (SONOS) bridge with a trivalent nitrogen, which constitutes an unusual native branching cross-link. In many proteins, the NOS switch contains a functionally essential lysine with direct roles in enzyme catalysis or binding of substrates, DNA or effectors, linking lysine chemistry and redox biology as a regulatory principle. NOS/SONOS switches are frequently found in proteins from human and plant pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and also in many human proteins with established roles in gene expression, redox signaling and homeostasis in physiological and pathophysiological conditions.