Debolina Hati

Date of Award

Fall 2022

Document Type

Open Access Dissertation

Department

Chemistry and Biochemistry

First Advisor

Caryn Outten

Second Advisor

F. Wayne Outten

Abstract

The trace metal iron is essential for the survival of almost all living organisms. It is a co-factor for many proteins involved in a variety of vital functions such as respiration, nitrogen fixation, and DNA biosynthesis, to name a few. Iron homeostasis in non-pathogenic fungi such as Schizosaccharomyces pombe has been extensively studied over the past few decades to better understand how intracellular iron is regulated under different environmental conditions. Iron regulation in S. pombe is controlled by two Fe-responsive transcriptional repressors known as Php4 and Fep1. Both rely on iron-sulfur (Fe-S) clusters as signals to balance iron acquisition, storage, and utilization in the cell. Furthermore, the cytosolic Fe-S cluster trafficking proteins Grx4 and Fra2 are required to control the activity of these proteins in response to iron. In this study, we tested the importance of conserved metal-binding residues in Fra2 and if mutating these residues abolishes or alters the ability of Fra2 to form a [2Fe-2S] binding complex with Grx4. We also used biochemical and spectroscopic tools to identify the type of iron cofactor harbored by Fep1 and characterize the metal-dependent interactions between S. pombe Fep1, Grx4, and Fra2. Our results show that His-66 and Cys-29 residues of Fra2 are involved in the formation of a [2Fe-2S] ligated Grx4-Fra2 heterocomplex, and are critical for the control of Fep1 activity in response to cellular iron status. Spectroscopic and biochemical analyses confirmed that Fep1 specifically binds a [2Fe-2S] cluster. We also report unidirectional [2Fe-2S] transfer from Fep1 to Grx4-Fra2, in the presence of GSH. Together, these results suggest that that Grx4 and Fra2 may control Fep1 function by facilitating removal of the Fe-S cluster from Fep1 under low iron conditions, leading to derepression of iron uptake genes. Since Fep1, Fra2, and Grx4 share homology with similar proteins in fungal pathogens, understanding iron regulation mechanisms at the cellular and molecular level in S. pombe may facilitate the design of new anti-fungal compounds to combat pathogenic fungi.

Rights

© 2022, Debolina Hati

Download

Included in

Chemistry Commons

Share

COinS