Redox-sensitive residue in the actin-binding interface of myosin

• Inhibition of nonmuscle myosin by H2O2 is due to oxidation of Met 394.
• This inhibition is specifically reversed by methionine sulfoxide reductase.
• In muscle, residue 394 is a Cys connected to the cardiomyopathy loop.
• The M394C mutant is reversibly inhibited by site-specific glutathionylation.
• This site is a potential redox sensor for modulation of motility and contractility.

We have examined the chemical and functional reversibility of oxidative modification in myosin. Redox regulation has emerged as a crucial modulator of protein function, with particular relevance to aging. We previously identified a single methionine residue in Dictyostelium discoideum (Dicty) myosin II (M394, near the myosin cardiomyopathy loop in the actin-binding interface) that is functionally sensitive to oxidation. We now show that oxidation of M394 is reversible by methionine sulfoxide reductase (Msr), restoring actin-activated ATPase activity. Sequence alignment reveals that M394 of Dicty myosin II is a cysteine residue in all human isoforms of skeletal and cardiac myosin. Using Dicty myosin II as a model for site-specific redox sensitivity of this Cys residue, the M394C mutant can be glutathionylated in vitro, resulting in reversible inhibition of actin-activated ATPase activity, with effects similar to those of methionine oxidation at this site. This work illustrates the potential for myosin to function as a redox sensor in both non-muscle and muscle cells, modulating motility/contractility in response to oxidative stress.