Crystal structures and calorimetry reveal catalytically relevant binding mode of coproporphyrin and coproheme in coproporphyrin ferrochelatase. (19th December 2019)

• Record Type: Journal Article
• Authors: Hofbauer, Stefan; Helm, Johannes; Obinger, Christian; Djinović‐Carugo, Kristina; Furtmüller, Paul G.
• Is Part Of: FEBS journal. Volume 287:Number 13(2020)
• Page Start: 2779
• Page End: 2796
• Publication Date: 2019-12-19
• Subjects: enzyme kinetics -- ferrochelatase -- heme biosynthesis -- X‐ray crystallography; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pathology, Molecular -- Periodicals; 572
• DOI: 10.1111/febs.15164
• Languages: English
• ISSNs: 1742-464X

Abstract

Abstract : Coproporphyrin ferrochelatases (CpfCs, EC 4.99.1.9 ) insert ferrous iron into coproporphyrin III yielding coproheme. CpfCs are utilized by prokaryotic, mainly monoderm (Gram‐positive) bacteria within the recently detected coproporphyrin‐dependent (CPD) heme biosynthesis pathway. Here, we present a comprehensive study on CpfC from Listeria monocytogenes ( Lm CpfC) including the first crystal structure of a coproheme‐bound CpfC. Comparison of crystal structures of apo‐ Lm CpfC and coproheme‐ Lm CpfC allowed identification of structural rearrangements and of amino acids involved in tetrapyrrole macrocycle and Fe 2+ binding. Differential scanning calorimetry of apo‐, coproporphyrin III‐, and coproheme‐ Lm CpfC underline the pronounced noncovalent interaction of both coproporphyrin and coproheme with the protein (Δ T m = 11 °C compared to apo‐ Lm CpfC), 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‐ Lm CpfC is presented as well as the kinetics of insertion of ferrous iron into coproporphyrin III‐ Lm CpfC that immediately leads to formation of ferric coproheme‐ Lm CpfC ( k cat / K M = 4.7 × 10 5 m −1 ·s −1 ). We compare the crystal structure of coproheme‐ Lm CpfC 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 CPD heme biosynthesis pathway. Enzyme: EC 4.99.1.9 Database: pdb‐codes of structural data in this work: 6RWV, 6SV3 . Abstract : Interactions of the coproheme propionate groups with coproporphyrin ferrochelatase in Listeria monocytogenes (Firmicutes) are identified by examination of the crystal structure of the complex. Propionates form H‐bonds or salt bridges to phylogenetically (within Firmicutes) conserved residues (presented in the corners). The proximal ligand of coproheme is a tyrosine and on the distal side a conserved histidine/glutamate pair is present (centre), which is responsible for binding of the divalent metal.