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We have identified C256 as the reactive cysteine residue that mediates OpdB inhibition by <jats:italic>N<\/jats:italic>\u2010ethylmaleimide and iodoacetic acid. Modeling studies suggest that C256 adducts occlude the P<jats:sub>1<\/jats:sub> substrate\u2010binding site, preventing substrate binding. We further demonstrate that C559 and C597 are responsible for the thiol\u2010enhancement of OpdB activity. 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