Investigation of Corynebacterium diphtheriae coproheme decarboxylases by resonance Raman spectroscopy and site-directed mutagenesis

Coproheme decarboxylases are important enzymes of the coproporphyrin-dependent heme biosynthesis pathway of Gram-positive bacteria (Firmicutes and Actinobacteria), necessary for survival and infection of hosts. These enzymes catalyze the oxidative decarboxylation of coproheme, converting the two propionates at positions 2 and 4 into vinyl groups via a stereospecific two-step process. This process first produces a three-propionate intermediate, the 2-monovinyl, 4-monopropionate deuteroheme, and finally heme b. Coproheme decarboxylases from several bacteria have been biochemically and spectroscopically characterized, with their structures solved and their catalytic processes analyzed in detail. Among several techniques, the combination of site-directed mutagenesis and resonance Raman spectroscopy has proven to be a powerful, synergistic approach that allows us to elucidate the roles of specific amino acids or residues in the protein's active site. In this review, we will focus on the resonance Raman data we have recently obtained on the coproheme decarboxylase from the actinobacterial Corynebacterium diphtheriae and selected site-directed variants. The results, together with UV-vis electronic absorption, X-ray crystallography, mass spectrometry, and molecular dynamics simulations, allowed us to elucidate the mechanism of the stepwise decarboxylation of coproheme to heme b and to rationalize the higher catalytic efficiency of this enzyme compared with coproheme decarboxylase from Firmicutes.