Engineering proximal vs. distal heme-NO coordination via dinitrosyl dynamics: implications for NO sensor design

Area of Science:

• Biochemistry
• Protein Engineering
• Sensor Technology

Background:

• Heme-based sensors utilize proximal vs. distal heme-NO coordination for selective gas response.
• Alcaligenes xylosoxidans cytochrome c' (AXCP) typically forms a distal 6-coordinate nitric oxide (NO) complex, followed by conversion to a proximal 5-coordinate NO product.

Purpose of the Study:

• To investigate the role of distal residue steric hindrance in AXCP's NO coordination pathway.
• To determine the mechanism controlling the formation of 6-coordinate vs. 5-coordinate NO products in heme-based sensors.

Main Methods:

• Site-directed mutagenesis of AXCP (e.g., Leu16 to Ala, Val, Ile, Phe).
• Crystallographic, spectroscopic, and kinetic measurements of engineered AXCP-NO complexes.
• Analysis of NO ligand kinetic parameters and steric effects.

Main Results:

• Mutations to smaller residues (Ala, Val, Ile) trapped the distal 6cNO complex.
• Mutations to larger residues (Leu, Phe) favored proximal 5cNO product formation.
• Increased distal steric hindrance distorted the Fe-N-O angle and heme propionate, but kinetic parameters were key for product outcome.

Conclusions:

• A 'balance of affinities' mechanism explains proximal 5cNO formation, relying on rapid distal NO release from a dinitrosyl precursor.
• This mechanism offers a novel strategy for NO sensing independent of weak Fe-His bonds.
• Findings provide a general approach for engineering selective gas responses in biological gas sensors for synthetic biology.