Sensing nitrite through a pseudoazurin-nitrite reductase electron transfer relay

Area of Science:

• Biochemistry
• Electrochemistry
• Microbiology

Background:

• Denitrification involves converting nitrite to nitric oxide via copper or haem enzymes.
• Efficient biosensor design necessitates quantitative assessment of this enzymatic turnover.
• Nitrite reductase from Alcaligenes faecalis possesses distinct copper centers and can be immobilized on gold electrodes.

Purpose of the Study:

• To quantitatively assess the enzymic turnover of nitrite reductase for biosensor development.
• To investigate methods for effectively coupling electron transfer to substrate catalytic turnover in immobilized enzymes.
• To develop an amperometric nitrite sensor utilizing enzyme-electrode interfaces.

Main Methods:

• Covalent tethering of Alcaligenes faecalis nitrite reductase to modified gold electrodes.
• Immobilization of a surface cysteine mutant of nitrite reductase on gold substrates.
• Utilizing redox partners like pseudoazurin or ruthenium hexammine as electron shuttles.
• Employing peptide-modified electrode surfaces for enzyme immobilization.

Main Results:

• Direct electron transfer was possible with covalently tethered enzyme configurations.
• Electron transfer coupling with substrate turnover was ineffective with a surface cysteine mutant on bare electrodes.
• The use of electron shuttles successfully coupled electron transfer to catalysis.
• This coupling enabled the development of an amperometric nitrite sensor.

Conclusions:

• Quantitative assessment of nitrite reductase enzymic turnover is crucial for biosensor design.
• Electron shuttles are effective in bridging electron transfer between immobilized enzymes and electrode surfaces.
• The developed amperometric nitrite sensor demonstrates the utility of enzyme-electrode interfaces coupled by electron conduits.