As established nearly a century ago, mechanoradicals originate from homolytic bond scission in polymers. The existence, nature and biological relevance of mechanoradicals in proteins, instead, are unknown. We here show that mechanical stress on collagen produces radicals and subsequently reactive oxygen species, essential biological signaling molecules. Electron-paramagnetic resonance (EPR) spectroscopy of stretched rat tail tendon, atomistic molecular dynamics simulations and quantum-chemical calculations show that the radicals form by bond scission in the direct vicinity of crosslinks in collagen. Radicals migrate to adjacent clusters of aromatic residues and stabilize on oxidized tyrosyl radicals, giving rise to a distinct EPR spectrum consistent with a stable dihydroxyphenylalanine (DOPA) radical. The protein mechanoradicals, as a yet undiscovered source of oxidative stress, finally convert into hydrogen peroxide. Our study suggests collagen I to have evolved as a radical sponge against mechano-oxidative damage and proposes a mechanism for exercise-induced oxidative stress and redox-mediated pathophysiological processes.

Ferroptosis is a type of cell death caused by radical-driven lipid peroxidation, leading to membrane damage and rupture. Here we show that enzymatically produced sulfane sulfur (S 0 ) species, specifically hydropersulfides, scavenge endogenously generated free radicals and, thereby, suppress lipid peroxidation and ferroptosis. By providing sulfur for S 0 biosynthesis, cysteine can support ferroptosis resistance independently of the canonical GPX4 pathway. Our results further suggest that hydropersulfides terminate radical chain reactions through the formation and self-recombination of perthiyl radicals. The autocatalytic regeneration of hydropersulfides may explain why low micromolar concentrations of persulfides suffice to produce potent cytoprotective effects on a background of millimolar concentrations of glutathione. We propose that increased S 0 biosynthesis is an adaptive cellular response to radical-driven lipid peroxidation, potentially representing a primordial radical protection system. NATURE CHEMiCAL BioLoGy | www.nature.com/naturechemicalbiology Articles NaTurE CHEmICal BIOlOGyprotect against ferroptosis independently of GPX4 by supplying sulfur to S 0 biosynthetic pathways. (3) The elevation of endogenous S 0 levels makes cells more resistant to LPO and ferroptosis, whereas the lowering of S 0 levels sensitizes cells to LPO and ferroptosis. (4) An increase of endogenous S 0 levels lowers endogenous radical load, and a decrease of endogenous S 0 levels increases endogenous radical load. (5) Exogenously supplied S 0 donors suppress ferroptosis in various cellular models. ( 6) Persulfides catalyze GSH-dependent radical scavenging in vitro, as they are regenerated via an autocatalytic cycle that couples radical reduction to glutathione oxidation through the formation and recombination of perthiyl radicals. Together, these results establish hydropersulfides as important radical scavengers and modulators of ferroptotic cell death. ResultsCystine uptake can inhibit ferroptosis independently of GPX4. It is well-understood that cellular cystine (Cys 2 ) uptake contributes to ferroptosis resistance. Inside the cell, Cys 2 is quickly reduced to Cys. Cys is needed for the synthesis of GSH, which, in turn, allows GPX4 to reduce lipid peroxides. However, it has been shown more recently that Cys is also a source of sulfur for S 0 species, including glutathione hydropersulfide and hydropolysulfides (GSS x H) 14,21 . Analyzing the cells used in this study, we found that about 1% of the total GSH pool is persulfidated (Extended Data Fig. 1a). We, therefore, asked if S 0 could contribute to ferroptosis resistance by suppressing LPO in a GPX4-independent manner (Fig. 1a). To address this question, we first assessed how endogenous S 0 levels change when cells are primed to undergo ferroptosis. Treatment of cells with the GPX4 inhibitor RSL3 or the lipophilic oxidant cumene hydroperoxide (CHP) increased endogenous S 0 levels (Fig. 1b and Extended Data Fig. 1b). Similarly, genetic deletion of floxed Gpx4 in Pfa1 cells 22 ...

Proteins are exposed to various mechanical loads that can lead to covalent bond scissions even before macroscopic failure occurs. Knowledge of these molecular breakages is important to understand mechanical properties of the protein. In regular molecular dynamics (MD) simulations, covalent bonds are predefined, and reactions cannot occur. Furthermore, such events rarely take place on MD time scales. Existing approaches that tackle this limitation either rely on computationally expensive quantum calculations (e.g., QM/MM) or complex bond order formalisms in force fields (e.g., ReaxFF). To circumvent these limitations, we present a new reactive kinetic Monte Carlo/molecular dynamics (KIMMDY) scheme. Here, bond rupture rates are calculated based on the interatomic distances in the MD simulation and then serve as an input for a kinetic Monte Carlo step. This easily scalable hybrid approach drastically increases the accessible time scales. Using this new technique, we investigate bond ruptures in a multimillion atom system of tensed collagen, a structural protein found in skin, bones, and tendons. Our findings show a clear concentration of bond scissions near chemical cross-links in collagen. We also examine subsequent dynamic relaxation steps. Our method exhibits only a minor slowdown compared to classical MD and is straightforwardly applicable to other complex (bio)­materials under load and related chemistries.