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

Coproheme decarboxylases are important enzymes of the coproporphyrin-dependent hemebiosynthesis pathway of Gram-positive bacteria (Firmicutes and Actinobacteria), necessary for survival andinfection of hosts. These enzymes catalyze the oxidative decarboxylation of coproheme, converting the twopropionates at positions 2 and 4 into vinyl groups via a stereospecific two-step process. This process firstproduces a three-propionate intermediate, the 2-monovinyl, 4-monopropionate deuteroheme, and finally hemeb. Coproheme decarboxylases from several bacteria have been biochemically and spectroscopicallycharacterized, with their structures solved and their catalytic processes analyzed in detail. Among severaltechniques, the combination of site-directed mutagenesis and resonance Raman spectroscopy has proven to bea powerful, synergistic approach that allows us to elucidate the roles of specific amino acids or residues in theprotein's active site. In this review, we will focus on the resonance Raman data we have recently obtained onthe coproheme decarboxylase from the actinobacterial Corynebacterium diphtheriae and selected site-directedvariants. 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 decarboxylationof coproheme to heme b and to rationalize the higher catalytic efficiency of this enzyme compared withcoproheme decarboxylase from Firmicutes.