Bromopyruvate as an affinity label for baker's yeast flavocytochrome b2. Stoichiometry of incorporation and localization on the peptide chain.

We have reported in a previous communication a kinetic study showing bromopyruvate to behave as an active-site-directed reagent for flavocytochrome b2. It is shown here that inactivation is accompanied by incorporation of 3 mol reagent/subunit of oxidized intact enzyme and 4 mol reagent/subunit nicked enzyme. Only one of the modifications is presumed to be responsible for activity loss. All labeled groups are found to be cysteines. Incubation of reduced nicked enzyme with bromopyruvate results in total protection of activity and loss of only one sulfhydryl group. A subsequent incubation in the presence of the competitive inhibitor sulfite leads to some more loss of non-essential groups. After these two pretreatments, incubation in the presence of bromo[2-14C]pyruvate results in incorporation of 1.2--1.5 mol reagent/subunit concomitant with the loss of about 0.8 active site. A study of the distribution of label between fragments alpha and beta has been carried out using gel electrophoresis and Sephadex filtration after selective proteolysis. It is shown that the active-site sulfhydryl group corresponds to one of the four cysteines situated in the last two thirds of fragment alpha. The structural and functional implications of these results is discussed.