Coproheme decarboxylase from Bacillus subtilis is required for bacterial growth and heme b biosynthesis under anaerobic conditions

Heme biosynthesis notably does not follow a universal pathway. Instead, different organisms utilize various routes of producing this essential molecule. The coproporphyrin-dependent (CPD) pathway is unique to Gram-positive bacteria. Given the ubiquity of Gram-positive pathogenic organisms, thorough research on its enzymatic steps is a prerequisite for the development of novel antibiotics. Here the focus lies on coproheme decarboxylase (ChdC, formerly HemQ), the terminal step of the pathway, catalyzing the transformation of Fe-coproporphyrin III (coproheme) to heme b by oxidative decarboxylation. In previous studies, hydrogen peroxide (H₂O₂) has been shown to act as a necessary co-substrate and electron acceptor for ChdC. However, H₂O₂, due to its cytotoxic effects, needs tight intracellular control and is a sub-optimal substrate in vivo, especially during anaerobic growth. To investigate a H₂O₂-free pathway for heme biosynthesis, knockout studies on Gram-positive model organism Bacillus subtilis have been performed. These reveal that ΔchdC strains exhibit heme auxotrophic behavior during aerobic and anaerobic growth, highlighting that ChdC has likely no anaerobic alternative. Free and protein bound FMN (in the form of flavodoxins YkuN and YkuP from B. subtilis) were subsequently characterized as alternative co-substrates. Their reaction with heterologously expressed ChdC from B. subtilis was characterized in different settings. By polarographic dioxygen level determination, liquid chromatography, mass spectrometry and time-resolved spectroscopy, these reactions were shown to be possible and promoted under anaerobic conditions and at elevated pH-values. Overall, the results presented in this study confirm the necessity and the capability of ChdC to react anaerobically in a Gram-positive model organism.