Measuring localized redox enzyme electron transfer in a live cell with conducting atomic force microscopy

Area of Science:

• Biochemistry
• Nanotechnology
• Microbiology

Background:

• Bacterial systems are crucial for energy, sensors, and industrial chemistry, but their molecular mechanisms remain unclear.
• Efficient bioengineered bacteria design necessitates a deep understanding of enzyme expression and activity at the cellular level.

Purpose of the Study:

• To develop and utilize a modified atomic force microscope (AFM) for detecting and analyzing redox-active enzyme activity on the surface of E. coli.
• To investigate the impact of electron transfer mediator proximity to the enzyme's NAD(+) binding site on electron transfer efficiency.

Main Methods:

• Engineered E. coli to display alcohol dehydrogenase II (ADHII) on their surface, with site-specific covalent attachment of a quinone electron transfer mediator.
• Employed an AFM with a gold-coated microwire cantilever serving as a working electrode in a three-electrode electrochemical cell.
• Performed single-cell electrochemical analysis by lifting individual bacteria for analysis in a redox-free buffer, comparing mutants with varying mediator-enzyme distances.

Main Results:

• Demonstrated the capability of a conducting AFM for single-cell electrochemical analysis of surface-displayed enzymes.
• Showed that electron transfer efficiency in redox-active proteins is enhanced when electron transfer mediators are positioned closer to the NAD(+) binding site.

Conclusions:

• The integrated conducting AFM system provides a powerful tool for detailed understanding of enzyme electron transfer processes.
• This approach is integral to advancing the development of efficiently engineered biosensors and biofuel cells.