Sulfide binding properties of truncated hemoglobins

Area of Science:

• Biochemistry
• Molecular Biology
• Protein-ligand interactions

Background:

• Truncated hemoglobins (trHbs) from Bacillus subtilis (Bs-trHb) and Thermobifida fusca (Tf-trHb) are known to bind ferric iron and hydrogen sulfide.
• Recombinant trHbs often contain residual iron-bound sulfide, necessitating detailed binding studies.
• Hydrogen sulfide (H2S) is a biologically relevant molecule involved in bacterial metabolic processes.

Purpose of the Study:

• To thermodynamically and kinetically characterize hydrogen sulfide binding to Bs-trHb and Tf-trHb.
• To elucidate the molecular mechanisms underlying the high affinity and slow dissociation of sulfide.
• To investigate the role of specific amino acid residues, particularly WG8, in sulfide binding stabilization.

Main Methods:

• Equilibrium and kinetic displacement experiments to determine sulfide binding parameters (affinity, on/off rates).
• Site-directed mutagenesis to alter specific distal residues (e.g., WG8 to Phe).
• Classical molecular dynamics simulations to model protein-ligand interactions.
• Resonance Raman spectroscopy to characterize the electronic state of the iron-sulfide complex.

Main Results:

• Both Bs-trHb and Tf-trHb exhibit high affinity for hydrogen sulfide (K ~10^6 M⁻¹ at pH 7.0), with affinity driven by slow sulfide release kinetics.
• The distal WG8 residue plays a crucial role in stabilizing bound sulfide, acting as a kinetic barrier to its release.
• A triple mutant (Tf-trHb with Phe at B10, G8, CD1) shows significantly enhanced affinity and faster sulfide release kinetics compared to the wild-type.
• Resonance Raman data suggest sulfide adducts are low-spin ferric iron derivatives with a characteristic Fe-S stretching band.

Conclusions:

• The high affinity of these truncated hemoglobins for hydrogen sulfide is primarily governed by the slow dissociation rate, influenced by specific distal residues like WG8.
• Mutational analysis and simulations confirm the critical role of WG8 in sulfide binding stabilization.
• The observed high affinity may have physiological relevance in bacterial thiol redox homeostasis and cysteine biosynthesis pathways.