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

DOI: 10.1111/febs.15164 DOI Help

Authors: Stefan Hofbauer (BOKU – University of Natural Resources and Life Sciences) , Johannes Helm (BOKU – University of Natural Resources and Life Sciences) , Christian Obinger (BOKU – University of Natural Resources and Life Sciences) , Kristina Djinović‐carugo (University of Vienna; University of Ljubljana) , Paul G. Furtmüller (BOKU – University of Natural Resources and Life Sciences)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: The Febs Journal

State: Published (Approved)
Published: December 2019

Abstract: Coproporphyrin ferrochelatases (CpfCs, EC insert ferrous iron into coproporphyrin III yielding coproheme. CpfCs are utilized by prokaryotic, mainly monoderm (Gram‐positive) bacteria within the recently detected coproporphyrin‐dependent heme biosynthesis pathway. Here we present a comprehensive study on CpfC from Listeria monocytogenes (LmCpfC) including the first crystal structure of a coproheme‐bound CpfC. Comparison of crystal structures of apo‐LmCpfC and coproheme‐LmCpfC allowed identification of structural rearrangements and of amino acids involved in tetrapyrrole macrocycle and Fe2+ binding. Differential scanning calorimetry of apo‐, coproporphyrin III‐ and coproheme‐LmCpfC underline the pronounced non‐covalent interaction of both coproporphyrin and coproheme with the protein (ΔTm = 11 °C compared to apo‐LmCpfC) which includes the propionates (p2, p4, p6, p7) and the amino acids Arg29, Arg45, Tyr46, Ser53 and Tyr124. Furthermore, the thermodynamics and kinetics of coproporphyrin III and coproheme binding to apo‐LmCpfC is presented as well as the kinetics of insertion of ferrous iron into coproporphyrin III‐LmCpfC that immediately leads to formation of ferric coproheme‐LmCpfC (kcat/KM = 4.7 × 105 M‐1 s‐1). We compare the crystal structure of coproheme‐LmCpfC with available structures of CpfCs with artificial tetrapyrrole macrocycles and discuss our data on substrate binding, iron insertion and substrate release in the context of the coproporphyrin‐dependent heme biosynthesis pathway.

Journal Keywords: heme biosynthesis; ferrochelatase; X‐ray crystallography; enzyme kinetics

Subject Areas: Chemistry

Instruments: I03-Macromolecular Crystallography

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