Molecular Dynamics Insights into the Effect of Glutathionylation at Different Sites on the Active Site of Human Ferrochelatase

Protoheme IX (Heme) is an essential cofactor for hemoglobin, with ferrochelatase (FECH) catalyzing the final step of its biosynthesis. While glutathione (GSH) has been shown to enhance FECH activity, the effects of site-specific glutathionylation at C323 and C395 remain elusive. Here, we applied molecular dynamics (MD) simulations to investigate the structural and functional consequences of four glutathionylation states– unglutathionylated (UG), GC323, GC395 and dual-modified (GC323[Formula: see text]GC395) — in both substrate-free and substrate-bound (protoporphyrin IX and Heme) forms. Our simulations revealed that glutathionylation at C395 enhances protoporphyrin IX binding and facilitates Heme release. Dual glutathionylation increased flexibility in the enzyme’s active site, suggesting a potential allosteric regulation mechanism. Key residues — R115, R164, H263, H341 and E343 — were identified as crucial for substrate coordination. We further confirmed the stability of FECH-substrate complexes through extended simulations. Binding free energy calculations via MM-PBSA supported the observed trends, while principal component analysis and dynamic cross-correlation analysis revealed distinct dynamic signatures associated with each modification state. Collectively, our findings provide new insights into the post-translational regulation of FECH activity by glutathionylation and suggest a mechanism by which redox status could modulate Heme biosynthesis.