Decarboxylation involving a ferryl, propionate, and a tyrosyl group in a radical relay yields heme b

The H2O2-dependent oxidative decarboxylation of coproheme III is the final step in the biosynthesis of heme b in many microbes. However, the coproheme decarboxylase reaction mechanism is unclear. The structure of the decarboxylase in complex with coproheme III suggested that the substrate iron, reactive propionates, and an active-site tyrosine convey a net 2e(-) /2H(+) from each propionate to an activated form of H2O2. Time-resolved EPR spectroscopy revealed that Tyr-145 formed a radical species within 30 s of the reaction of the enzyme-coproheme complex with H2O2. This radical disappeared over the next 270 s, consistent with a catalytic intermediate. Use of the harderoheme III intermediate as substrate or substitutions of redox-active side chains (W198F, W157F, or Y113S) did not strongly affect the appearance or intensity of the radical spectrum measured 30 s after initiating the reaction with H2O2, nor did it change the similar to 270 s required for the radical signal to recede to <= 10% of its initial intensity. These results suggested Tyr-145 as the site of a catalytic radical involved in decarboxylating both propionates. Tyr-145(center dot) was accompanied by partial loss of the initially present Fe(III) EPR signal intensity, consistent with the possible formation of Fe(IV) = O. Site-specifically deuterated coproheme gave rise to a kinetic isotope effect of similar to 2 on the decarboxylation rate constant, indicating that cleavage of the propionate C beta-H bond was partly rate-limiting. The inferred mechanism requires two consecutive hydrogen atom transfers, first from Tyr-145 to the substrate Fe/H2O2 intermediate and then from the propionate C beta-H to Tyr-145(center dot).