HmuT in the Heme Uptake Pathway of Corynebacterium diphtheriae: stability and function

Strategies to use heme as a source of iron are key to the survival and virulence of many bacteria; inhibition of iron uptake pathways may be a new strategy to prevent bacterial infection. Corynebacterium diphtheriae is a Gram-positive, pathogenic bacterium that is the causative agent of diphtheria It utilizes proteins in heme uptake pathways to obtain required iron for survival and virulence. One uptake pathway involves an ABC-type transporter encoded by the hmuTUV genes. We analyze the role of HmuT, the protein that donates heme to the ABC transporter. We hypothesize that certain residues in the heme pocket, in addition to the direct heme binding ligands, control heme binding and release in HmuT. Sequence alignment with other heme-binding proteins and I-TASSER homology modeling revealed the following possible essential residues: H136, Y235, Y272, Y349, R237 and M292. Site-directed mutagenesis was used to create alanine mutants for these residues. Mutants studied include H136A, Y235A, Y272A, and M292A. UV-visible spectroscopy was used to compare spectral signatures of the WT to the mutants. In addition, chemical and thermal unfolding experiments were performed to assess the contribution of each residue to heme binding. We have shown that H136 and Y235 are axial ligands to the heme while M292 appears to buttress the axial tyrosine. R237 is a Hbonding partner to Y235. Y272, and Y349 in the heme pocket as well, also affects heme binding. Understanding heme proteins create a possible new strategy to prevent bacterial infection by inhibiting iron uptake pathways. Introduction Many living organisms, including pathogenic bacteria, require iron for survival (1). In human, most of the iron is found in heme (2). The tetrapyrrole ring of heme allows usage of the iron as a cofactor for enzymes in activities such as oxygen transport, cellular respiration and signal transduction. Many bacteria have developed pathways to take up heme and degrade the tetrapyrrole ring to release the iron. Corynebacterium diphtheriae, a Gram-positive pathogenic bacterium that utilizes heme uptake pathways to obtain required iron from it host (3). It is the causative agent of diphtheria, a prevalent upper respiratory tract disease that has a high mortality rate in areas with low vaccine coverage (4). C. diphtheriae requires iron for survival and virulence (5-9). Prior study of C. diphtheriae showed that the hmu gene cluster encodes a transmembrane protein, the hmuTUV ABC transporter, and surface proteins HtaA and HtaC (10). Other Corynebacterium species share the hmuTUV genes sequences. The deletion of the hmuTUV, HtaA or the entire hmu gene cluster resulted in reduced call growth, indicating that the hmu gene