Hemerythrin is a respiratory protein found in the erythrocytes of certain marine invertebrates. Structures have been obtained by x-ray crystallography for hemerythrins from Phascolopsis gouldii (P. gouldii) and Themiste dyscritum (T. dyscritum). Since many proteins are inactivated upon binding heavy metals such as mercury, an investigation of mercury-protein interactions in this protein was undertaken. In order to understand the nature of heavy-metal protein interactions in T. dyscritum it was necessary to identify the metalloprotein ligands since neither the liganding amino acid residues nor the geometry of the mercury complexes could be identified in the crystallographic studies.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, one of the top ten causes of death worldwide. One of the genes upregulated in Mtb during macrophage infection is rv2633c, but the structure and function of its gene product remain unknown. Preliminary research has indicated that Rv2633c is a hemerythrin-like protein that exhibits catalase activity and binds two iron atoms using an HHE domain. Additionally, Rv2633c appears to exist as a dimer. The purpose of this project is to identify specific residues outside of the HHE domain that contribute to the protein's iron-binding ability and/or catalase activity, and to determine whether residues on the C terminus are required for dimerization. Conserved residues D37, E42, and E95 were selected due to their proximity in the amino acid sequence to the HHE domain. Each residue was mutated to alanine using site-directed mutagenesis and the mutations were confirmed using Sanger sequencing. The E95A mutant and the C-terminal truncation mutant were expressed in Escherichia coli using the T7 expression system and purified using affinity chromatography. While wild-type Rv2633c eluted as a soluble protein, the C-terminal truncation mutant was not soluble, indicating that the C terminus may be required for Rv2633c folding. The E95A mutant eluted as a soluble protein, but may have lower iron content than wild-type Rv2633c, indicating that this glutamic acid residue could contribute to iron-binding, despite being outside the HHE domain.

Hemerythrin-like protein Rv2633c is a small 18 kDa protein that is expressed in Mycobacterium tuberculosis (Mtb). Sequence analysis of Rv2633c predicts the presence of a hemerythrin-like domain, which binds dioxygen using a µ-oxo-bridge (Fe-O-Fe), rather than a heme group. Though it is noticeably upregulated during macrophage infection and during in vitro acidification, the role of Rv2633c in Mtb survival has yet to be elucidated. This project aims to characterize the function of Rv2633c by studying the in vitro response of the recombinant protein to conditions present in the macrophage lysosome, such as reduced oxygen levels or the presence of reactive oxygen species. UV-visible spectroscopy is used to observe these changes, as the spectrum shows a characteristic peak at 330 nm that likely corresponds to the diiron cofactor in its native state. Our results show this spectrum shifts in response to hydrogen peroxide addition, showing the proposed environmental conditions can affect the active site. Bioinformatics techniques, such as the 3D modeling program SWISS-MODEL, have been used to hypothesize possible structure and function. Determining the function of Rv2633c may help explain how Mtb so readily evades the human immune system to reside in the macrophage.