Pyruvate decarboxylase (E1) catalyzes the first two reactions of the four involved in oxidative decarboxylation of pyruvate by the pyruvate dehydrogenase (PDH) multienzyme complex. It requires thiamin diphosphate to bring about the decarboxylation of pyruvate, which is followed by the reductive acetylation of a lipoyl group covalently bound to the N(6) amino group of a lysine residue in the second catalytic component, a dihydrolipoyl acetyltransferase (E2). Replacement of two histidine residues in the E1alpha and E1beta chains of the heterotetrameric E1 (alpha(2)beta(2)) component of the PDH complex of Bacillus stearothermophilus, considered possible proton donors at the active site, was carried out. Subsequent characterization of the mutants permitted different roles to be assigned to these two particular residues in the reaction catalyzed by E1: E1alpha His271 to stabilize the dianion formed during decarboxylation of the 2-oxo acid and E1beta His128 to provide the proton required to protonate the incoming dithiolane ring in the subsequent reductive acetylation of the lipoyl goup. On the basis of these and other results from a separate investigation into the roles of individual residues in a loop region in the E1alpha chain close to the active site of E1 [Fries, M., Chauhan, H. J., Domingo, G. J., Jung, H., and Perham, R. N. (2002) Eur. J. Biochem. 270, 861-870] together with work from other laboratories, a detailed mechanism for the E1 reaction can be formulated.