Spectroscopic studies on HasA from Yersinia pseudotuberculosis.

Area of Science:

• Microbiology
• Biochemistry
• Structural Biology

Background:

• Heme acquisition system A (HasA) functions as a hemophore in Gram-negative pathogens.
• HasA facilitates the uptake of heme, an essential iron source for bacterial survival.
• Understanding HasA's mechanism is vital for developing novel antimicrobial strategies.

Purpose of the Study:

• To investigate the heme binding and transfer mechanisms of HasA from Yersinia pseudotuberculosis (HasAyp).
• To compare the heme coordination properties of HasAyp with HasA from Pseudomonas aeruginosa (HasApa).
• To elucidate the role of specific amino acid residues, particularly Tyr-75 and Gln-32, in HasAyp function.

Main Methods:

• Spectroscopic studies (e.g., resonance Raman spectroscopy) were employed to analyze heme coordination.
• Site-directed mutagenesis was used to create specific amino acid substitutions (e.g., Y75A, Gln-32 to alanine).
• Hemin uptake and heme transfer assays were performed using wild-type and mutant HasAyp.

Main Results:

• Spectroscopic data suggest that only Tyr-75 coordinates ferric heme iron in HasAyp, unlike HasApa which involves His-32.
• Gln-32 is not an axial ligand for heme iron in HasAyp.
• The Y75A mutation impaired but did not abolish hemin uptake, indicating a role for hydrophobic interactions.
• Tyr-75 coordination is critical for efficient heme transfer from hemoglobin (Hb) to HasAyp.
• HasAyp uniquely binds ferrous heme iron, with Tyr-75 becoming protonated in the ferrous-carbon monoxide complex.

Conclusions:

• Tyr-75 is essential for ferric heme iron coordination and transfer from Hb by HasAyp.
• Hydrophobic interactions contribute to heme acquisition by HasAyp.
• HasAyp's ability to bind ferrous heme may confer an advantage to Y. pseudotuberculosis, a facultative anaerobe.