In oxygenic phototrophs, chlorophylls, hemes, and bilins are synthesized by a common branched pathway. Given the phototoxic nature of tetrapyrroles, this pathway must be tightly regulated, and an important regulatory role is attributed to magnesium chelatase enzyme at the branching between the heme and chlorophyll pathway. Gun4 is a porphyrin-binding protein known to stimulate in vitro the magnesium chelatase activity, but how the Gun4-porphyrin complex acts in the cell was unknown. To address this issue, we first performed simulations to determine the porphyrin-docking mechanism to the cyanobacterial Gun4 structure. After correcting crystallographic loop contacts, we determined the binding site for magnesium protoporphyrin IX. Molecular modeling revealed that the orientation of α6/α7 loop is critical for the binding, and the magnesium ion held within the porphyrin is coordinated by Asn-211 residue. We also identified the basis for stronger binding in the Gun4-1 variant and for weaker binding in the W192A mutant. The W192A-Gun4 was further characterized in magnesium chelatase assay showing that tight porphyrin binding in Gun4 facilitates its interaction with the magnesium chelatase ChlH subunit. Finally, we introduced the W192A mutation into cells and show that the Gun4-porphyrin complex is important for the accumulation of ChlH and for channeling metabolites into the chlorophyll biosynthetic pathway.

Department of Molecular Biology and Biotechnology University of Sheffield Sheffield S10 2TN United Kingdom

Institute of Microbiology Academy of Sciences 37981 Třeboň Czech Republic

Institute of Microbiology Academy of Sciences 37981 Třeboň Czech Republic; Faculty of Science University of South Bohemia 370 05 České Budějovice Czech Republic

Joint Barcelona Supercomputing Center Centre for Genomic Regulation Institute for Research in Biomedicine Research Program Carrer de Jordi Girona 29 08034 Barcelona Spain

Joint Barcelona Supercomputing Center Centre for Genomic Regulation Institute for Research in Biomedicine Research Program Carrer de Jordi Girona 29 08034 Barcelona Spain; Institució Catalana de Recerca i Estudis Avançats 08010 Barcelona Spain

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Evolution of Ycf54-independent chlorophyll biosynthesis in cyanobacteria

A Photosynthesis-Specific Rubredoxin-Like Protein Is Required for Efficient Association of the D1 and D2 Proteins during the Initial Steps of Photosystem II Assembly

The antenna-like domain of the cyanobacterial ferrochelatase can bind chlorophyll and carotenoids in an energy-dissipative configuration

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PDB
1Y6I, 1Z3X