Oxidative stress is a common challenge to mitochondrial function where reactive oxygen species are capable of significant organelle damage. The generation of mitochondrial reactive oxygen species occurs in the inner membrane and matrix compartments as a consequence of subunit function in the electron transport chain and citric acid cycle， respectively. Maintenance of mitochondrial proteostasis and stress response is facilitated by compartmentalized proteases that couple the energy of ATP hydrolysis to unfolding and the regulated removal of damaged， misfolded， or aggregated proteins. The mitochondrial protease YME1L functions in the maintenance of proteostasis in the intermembrane space. YME1L is an inner membrane-anchored hexameric protease with distinct N-terminal， transmembrane， AAA+ (ATPases associated with various cellular activities)， and C-terminal M41 zinc-dependent protease domains. The effect of oxidative stress on enzymes such as YME1L tasked with maintaining proteostasis is currently unclear. We report here that recombinant YME1L undergoes a reversible conformational change in response to oxidative stress that involves the interaction of one hydrogen peroxide molecule per YME1L monomer with affinities equal to 31 ± 2 and 26 ± 1 mM for conditions lacking or including nucleotide， respectively. Our data also reveal that oxidative stress does not significantly impact nucleotide binding equilibria， but does stimulate a 2-fold increase in the rate constant for high-affinity ATP binding from (8.9 ± 0.2) × 10 M s to (1.5 ± 0.1) × 10 M s. Taken together， these data may suggest a mechanism for the regulated processing of YME1L by other inner membrane proteases such as OMA1.