# Impact of Phosphorylation at Various Sites on the Active Pocket of Human Ferrochelatase: Insights from Molecular Dynamics Simulations

## TL;DR

This study uses simulations to explore how phosphorylation at different sites affects the function of ferrochelatase, a key enzyme in heme production.

## Contribution

The study identifies a new phosphorylation site (T218) and reveals collaborative effects of phosphorylation on ferrochelatase activity.

## Key findings

- Phosphorylation at T116 and T218 together lowers binding free energy with PPIX, indicating stronger binding.
- The PT116 + PT218 state increases Heme release by having higher binding free energy compared to unphosphorylated FECH.
- Phosphorylation alters FECH dynamics and substrate interactions, as shown through multiple analysis methods.

## Abstract

Ferrochelatase (FECH) is the terminal enzyme in human heme biosynthesis, catalyzing the insertion of ferrous iron into protoporphyrin IX (PPIX) to form protoheme IX (Heme). Phosphorylation increases the activity of FECH, and it has been confirmed that the activity of FECH phosphorylated at T116 increases. However, it remains unclear whether the T116 site and other potential phosphorylation modification sites collaboratively regulate the activity of FECH. In this study, we identified a new phosphorylation site, T218, and explored the allosteric effects of unphosphorylated (UP), PT116, PT218, and PT116 + PT218 states on FECH in the presence and absence of substrates (PPIX and Heme) using molecular dynamics (MD) simulations. Binding free energies were evaluated with the MM/PBSA method. Our findings indicate that the PT116 + PT218 state exhibits the lowest binding free energy with PPIX, suggesting the strongest binding affinity. Additionally, this state showed a higher binding free energy with Heme compared to UP, which facilitates Heme release. Moreover, employing multiple analysis methods, including free energy landscape (FEL), principal component analysis (PCA), dynamic cross-correlation matrix (DCCM), and hydrogen bond interaction analysis, we demonstrated that phosphorylation significantly affects the dynamic behavior and binding patterns of substrates to FECH. Insights from this study provide valuable theoretical guidance for treating conditions related to disrupted heme metabolism, such as various porphyrias and iron-related disorders.