Uroporphyrinogen III decarboxylase catalyzes the fifth step in heme biosynthesis: the elimination of carboxyl groups from the four acetate side chains Of Uroporphyrinogen III to yield coproporphyrinogen III. The enzyme acts by successively protonating each of the four pyrrole rings present in the substrate, thereby allowing decarboxylation of their side chains, but the identity of the proton donors has not been established yet. Tyr164 has been suggested as a proton donor, and Asp86 has been proposed to act either as a proton donor or as an interinediate-stabilizing residue. We have performed density-functional calculations to study this reaction mechanism, and found that the rate-limiting step is substrate protonation, rather than decarboxylation. Surprisingly, whereas Tyr164 is unable to protonate the substrate, this protonation can be effected by a nearby arginine residue (Arg37), with a free energy barrier of 21.4 kcal center dot mol(-1), in remarkable agreement with the experimental value of 19.5 kcal center dot mol(-1). The central positioning of this residue in close proximity to all four pyrrole rings in the substrate may play a key role in the sequential activation of each of these moieties.