Heme Binding by Corynebacterium diphtheriae HmuT: Function and Heme Environment

Area of Science:

• Microbiology
• Biochemistry
• Structural Biology

Background:

• Corynebacterium diphtheriae requires heme for growth and utilizes a heme uptake (hmu) pathway.
• Heme is delivered to the ABC transporter HmuUV by the protein HmuT.
• Understanding HmuT's heme binding is critical for elucidating the heme uptake mechanism.

Purpose of the Study:

• To determine the axial heme ligands in ferric HmuT.
• To investigate the roles of conserved heme pocket residues (H136, Y235, M292) in heme binding and transport.
• To characterize the heme environment within HmuT.

Main Methods:

• Site-directed mutagenesis of HmuT (H136A, Y235A, M292A).
• Spectroscopic analyses: UV-visible, resonance Raman, and magnetic circular dichroism.
• Redox potential measurements and heme-ligand complexation (FeCO, FeF).
• Hemoglobin utilization assays.

Main Results:

• Spectroscopic data indicate Histidine-136 and Tyrosine-235 are the axial ligands in ferric HmuT.
• Mutations H136A and Y235A affect heme reduction potentials, consistent with ligand assignments.
• FeCO complex analysis provides further evidence for axial ligand identity and heme pocket environment.
• Y235A mutation significantly impairs heme uptake, highlighting its essential role.

Conclusions:

• Histidine-136 and Tyrosine-235 serve as the axial ligands for heme in HmuT.
• Tyrosine-235 is essential for HmuT function in heme uptake.
• The study provides insights into the heme binding site and its role in heme transport by C. diphtheriae.