NO binding induced conformational changes in a truncated hemoglobin from Mycobacterium tuberculosis

Area of Science:

• Biochemistry
• Spectroscopy
• Protein Dynamics

Background:

• Hemoglobin N (HbN) from Mycobacterium tuberculosis is a non-symbiotic hemoglobin with potential regulatory roles.
• Nitric oxide (NO) is a crucial signaling molecule whose interaction with hemoglobins is of significant biological interest.
• Understanding the structural basis of NO binding in HbN can elucidate its functional mechanisms.

Purpose of the Study:

• To investigate the interaction between the B10 Tyr residue and heme-bound NO in wild-type HbN and a B10 Tyr mutant.
• To characterize the conformational changes induced by NO binding in HbN using resonance Raman spectroscopy.
• To explore the functional implications of these interactions and conformational changes on HbN's properties.

Main Methods:

• Resonance Raman spectroscopy with Soret and UV excitation was employed.
• Spectra of wild-type HbN and the B10 Tyr --> Phe mutant were compared in their NO-bound ferric states.
• Difference spectroscopy was used to isolate the spectral contributions of the B10 Tyr residue.

Main Results:

• The Fe-NO stretching and bending modes were assigned for wild-type HbN.
• Mutation of B10 Tyr to Phe altered Fe-NO and N-O stretching frequencies, suggesting H-bonding interactions.
• UV Raman analysis confirmed direct interaction of B10 Tyr with heme-bound NO and revealed NO-induced conformational changes propagating through the protein.

Conclusions:

• The B10 Tyr residue in HbN directly interacts with heme-bound NO, influencing its orientation.
• NO binding to HbN triggers significant, large-scale conformational rearrangements.
• These conformational changes likely play a role in modulating HbN's ligand-binding affinity and chemical reactivity.