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

Alfonta L, Meckes B, Amir L, Schlesinger O, Ramachandran S, Lal R - Anal. Chem. (2014)

Bottom Line: A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII.An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed.Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, ‡Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , P.O. Box 653, Beer-Sheva, 84105, Israel.

ABSTRACT
Bacterial systems are being extensively studied and modified for energy, sensors, and industrial chemistry; yet, their molecular scale structure and activity are poorly understood. Designing efficient bioengineered bacteria requires cellular understanding of enzyme expression and activity. An atomic force microscope (AFM) was modified to detect and analyze the activity of redox active enzymes expressed on the surface of E. coli. An insulated gold-coated metal microwire with only the tip conducting was used as an AFM cantilever and a working electrode in a three-electrode electrochemical cell. Bacteria were engineered such that alcohol dehydrogenase II (ADHII) was surface displayed. A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII. The AFM probe was used to lift a single bacterium off the surface for electrochemical analysis in a redox-free buffer. An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed. Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site. This study suggests that an integrated conducting AFM used for single cell electrochemical analysis would allow detailed understanding of enzyme electron transfer processes to electrodes, the processes integral to creating efficiently engineered biosensors and biofuel cells.

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SchematicDescription of the EC-AFM SetupThe materials for the electrodes,reference (RE), working (WE), and counter (CE), were Ag/AgCl, Au,and platinum, respectively.
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sch1: SchematicDescription of the EC-AFM SetupThe materials for the electrodes,reference (RE), working (WE), and counter (CE), were Ag/AgCl, Au,and platinum, respectively.


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

Alfonta L, Meckes B, Amir L, Schlesinger O, Ramachandran S, Lal R - Anal. Chem. (2014)

SchematicDescription of the EC-AFM SetupThe materials for the electrodes,reference (RE), working (WE), and counter (CE), were Ag/AgCl, Au,and platinum, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4215851&req=5

sch1: SchematicDescription of the EC-AFM SetupThe materials for the electrodes,reference (RE), working (WE), and counter (CE), were Ag/AgCl, Au,and platinum, respectively.
Bottom Line: A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII.An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed.Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site.

View Article: PubMed Central - PubMed

Affiliation: Department of Life Sciences, ‡Ilse Katz Institute for Nanoscale Science and Technology, Ben-Gurion University of the Negev , P.O. Box 653, Beer-Sheva, 84105, Israel.

ABSTRACT
Bacterial systems are being extensively studied and modified for energy, sensors, and industrial chemistry; yet, their molecular scale structure and activity are poorly understood. Designing efficient bioengineered bacteria requires cellular understanding of enzyme expression and activity. An atomic force microscope (AFM) was modified to detect and analyze the activity of redox active enzymes expressed on the surface of E. coli. An insulated gold-coated metal microwire with only the tip conducting was used as an AFM cantilever and a working electrode in a three-electrode electrochemical cell. Bacteria were engineered such that alcohol dehydrogenase II (ADHII) was surface displayed. A quinone, an electron transfer mediator, was covalently attached site specifically to the displayed ADHII. The AFM probe was used to lift a single bacterium off the surface for electrochemical analysis in a redox-free buffer. An electrochemical comparison between two quinone containing mutants with different distances from the NAD(+) binding site in alcohol dehydrogenase II was performed. Electron transfer in redox active proteins showed increased efficiency when mediators are present closer to the NAD(+) binding site. This study suggests that an integrated conducting AFM used for single cell electrochemical analysis would allow detailed understanding of enzyme electron transfer processes to electrodes, the processes integral to creating efficiently engineered biosensors and biofuel cells.

Show MeSH