Spectroscopic evidence of the effect of hydrogen peroxide excess on the coproheme decarboxylase from actinobacterial Corynebacterium diphtheriae .

The actinobacterial coproheme decarboxylase from Corynebacterium diphtheriae catalyzes the final reaction to generate heme b via the "coproporphyrin-dependent" heme biosynthesis pathway in the presence of hydrogen peroxide. The enzyme has a high reactivity toward H 2 O 2 used for the catalytic reaction and in the presence of an excess of H 2 O 2 new species are generated. Resonance Raman data, together with electronic absorption spectroscopy and mass spectrometry, indicate that an excess of hydrogen peroxide for both the substrate (coproheme) and product (heme b ) complexes of this enzyme causes a porphyrin hydroxylation of ring C or D, which is compatible with the formation of an iron chlorin-type heme d species. A similar effect has been previously observed for other heme-containing proteins, but this is the first time that a similar mechanism is reported for a coproheme enzyme. The hydroxylation determines a symmetry lowering of the porphyrin macrocycle, which causes the activation of A 2g modes upon Soret excitation with a significant change in their polarization ratios, the enhancement and splitting into two components of many E u bands, and an intensity decrease of the non-totally symmetric modes B 1g , which become polarized. This latter effect is clearly observed for the isolated ν 10 mode upon either Soret or Q-band excitations. The distal His118 is shown to be an absolute requirement for the conversion to heme d . This residue also plays an important role in the oxidative decarboxylation, because it acts as a base for deprotonation and subsequent heterolytic cleavage of hydrogen peroxide.