Soluble succinate dehydrogenase prepared by butanol extraction reacts with N-ethylmaleimide according to first-order kinetics with respect to both remaining active enzyme and the inhibitor concentration. Binding of the sulfhydryl groups of the enzyme prevents its alkylation by Nethylmaleimide and inhibition by oxaloacetate. A kinetic analysis of the inactivation by alkylating reagent in the presence of succinate or malonate suggests that N-ethylmaleimide acts as a sitcdirected inhibitor. The apparent first-order rate constant of alkylation increases between pH 5.8 and 7.8 indicating a pK, value for the enzyme sulfhydryl group equal to 7.0 at 22 "C in 50 mM Trissulfate buffer. Certain anions (phosphate, citrate, maleate and acetate) decrease the reactivity of the enzyme towards the alkylating reagent. Succinate/phenazine methosulfate reductase activity measured in the presence of a saturating concentration of succinate shows the same pH-dependence as the alkylation rate by N-ethylmaleimide. The mechanism of the first step of succinate oxidation. including a nucleophilic attack of substrate by the active-site sulfhydryl group, is discussed.

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