The coproporphyrin ferrochelatase of Staphylococcus aureus: mechanistic insights into a regulatory iron-binding site

Abstract

The majority of characterised ferrochelatase enzymes catalyse the final step of classical haem synthesis, inserting ferrous iron into protoporphyrin IX. However, for the recently discovered coproporphyrin-dependent pathway, ferrochelatase catalyses the penultimate reaction where ferrous iron is inserted into coproporphyrin III. Ferrochelatase enzymes from the bacterial phyla Firmicutes and Actinobacteria have previously been shown to insert iron into coproporphyrin, and those from Bacillus subtilis and Staphylococcus aureus are known to be inhibited by elevated iron concentrations. The work herein reports a Km (coproporphyrin III) for S. aureus ferrochelatase of 1.5 µM and it is shown that elevating the iron concentration increases the Km for coproporphyrin III, providing a potential explanation for the observed iron-mediated substrate inhibition. Together, structural modelling, site-directed mutagenesis, and kinetic analyses confirm residue Glu271 as being essential for the binding of iron to the inhibitory regulatory site on S. aureus ferrochelatase, providing a molecular explanation for the observed substrate inhibition patterns. This work therefore has implications for how haem biosynthesis in S. aureus is regulated by iron availability.

Crystal structures and calorimetry reveal catalytically relevant binding mode of coproporphyrin and coproheme in coproporphyrin ferrochelatase

“…Steady-state kinetic parameters of the two-substrate reaction were derived for ferrous iron, which was varied in concentration, whereas coproporphyrin III was under saturated conditions. The catalytic specificity is comparable to the one previously reported for the highly similar CpfC from S. aureus (SaCpfC) [15]. The determined K M value is 0.28 µM for Fe(II), and k cat is 7.9 min À1 , yielding a catalytic specificity of 4.7 9 10 5 M -1 Ás À1 .…”

“…The determined K M value is 0.28 µM for Fe(II), and k cat is 7.9 min À1 , yielding a catalytic specificity of 4.7 9 10 5 M -1 Ás À1 . Substrate inhibition was evident at Fe(II) concentrations higher than 0.8 µM, as for SaCpfC [15] (Fig. 4E).…”

“…Architecture of the active site of ferrochelatases is described by the proximal Tyr12 and the distal His182/Glu263 pair (Fig. 8A), which has been shown to be the metal binding site of CpfCs [8,15]. In the given structure, Glu263 was best modeled in a double conformation, forming an H-bond in one conformation with His182; this reflects the flexibility of this residue (Fig.…”

Blue Light Disinfection in Hospital Infection Control: Advantages, Drawbacks, and Pitfalls

“…These iron-free porphyrins have two possible origins. They are either synthetized by bacteria as a by-product of heme biosynthesis or they arise as residuum of porphyrins that had their heme taken by the bacteria [114,115,116,117,118].…”

“…The same research group also described the presence of protoporphyrin IX not only in P. aeruginosa, but also in A. baumannii [114]. As for S. aureus and C. albicans , uroporphyrin and coproporphyrin, and flavins, respectively, were the almost exclusively produced photo-sensitizers [117,118,120]. The inactivation of H. pylori was also found to be related with coproporphyrin and protoporphyrin IX [111,121].…”