Modeling proline ligation in the heme-dependent CO sensor, CooA, using small-molecule analogs

Area of Science:

• Biochemistry and Molecular Biology
• Protein-ligand interactions
• Transcription factor regulation

Background:

• CooA from Rhodospirillum rubrum is a unique CO-activated transcription factor utilizing proline as a heme ligand.
• Proline acts as a heme ligand in both Fe(III) and Fe(II) states, with cysteinate and histidine as the sixth ligands, respectively.
• CO binding to Fe(II) CooA displaces the proline ligand, activating the protein for its regulatory function.

Purpose of the Study:

• To investigate the role of proline's weak metal-ligand bond in selective CO binding and protein activation.
• To understand how CooA's heme pocket accommodates the proline ligand and facilitates CO sensing.
• To elucidate the structural and electronic factors governing the proline-heme interaction and its displacement by CO.

Main Methods:

• Binding affinity measurements of proline and histidine analogs (pyrrolidine, 2-methylpyrrolidine, imidazole) in CooA and myoglobin variants.
• Structural studies using X-ray crystallography of model metalloporphyrins.
• Density functional theory (DFT) computational studies to analyze electronic and steric effects.

Main Results:

• CooA's heme pocket is specifically adapted to accommodate the bulky proline ligand.
• Steric hindrance at the C-2 position of the pyrrolidine ring significantly weakens the Fe-ligand bond.
• The high pKa of proline and steric factors contribute to its selective displacement by CO.

Conclusions:

• CooA employs a weak proline-heme bond, weakened by steric hindrance, to ensure selective CO binding.
• This mechanism allows for efficient CO sensing and activation of the transcription factor.
• The findings provide a deeper understanding of how CooA utilizes proline for precise control of gene expression in response to CO.