Limits...
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.

Show MeSH
Cyclic voltammogramsof the different mutants; lavender coloredline: mutant V66Az; rose colored line: mutant D314Az; lime green coloredline: background measurement with an unmodified working electrode.Scan rates were 0.05 V s–1; reference electrode:Ag/AgCl wire.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4215851&req=5

fig4: Cyclic voltammogramsof the different mutants; lavender coloredline: mutant V66Az; rose colored line: mutant D314Az; lime green coloredline: background measurement with an unmodified working electrode.Scan rates were 0.05 V s–1; reference electrode:Ag/AgCl wire.

Mentions: Figure 4 shows the cyclic voltammograms collected usinga mutant V66Az modified AFM tip (lavender colored line), a mutantD314Az modified tip (rose colored line), and a background measurementof the unmodified working electrode (lime green colored line) collectedat a scan rate of 0.05 V s–1. When studying the electrochemicalproperties of linker 1, based on eq 1, plotted Ip values against scan ratesdetermined a surface coverage of Γ = 2.3 × 10–10 mol·cm–2. However, surface coverage valuescalculated for the surface modified bacteria containing linker 1 bound to the surface of the electrode yielded values ofΓo = 8.0 × 10–11 mol·cm–2 and ΓR = 1.0 × 10–11 mol·cm–2 for mutant V66Az, oxidized and reducedforms, respectively, whereas the values of surface coverage for mutantD314Az were Γo = 2.7 × 10–11 mol·cm–2 and ΓR = 8.0 ×10–12 mol·cm–2, for the oxidizedand reduced forms. The much lower conversion values for the mutants, compared to thosemeasured for linker 1 by itself, suggest that there isan additional process that is involved, namely, the redox reactionof NADH. Taking into account the redox potential measured for mutantD314Az and the fact that it is identical to the redox potential oflinker 1, but with much lower reversibility, it is suggestedthat the degree of mediation of ET between NAD+ and thequinone containing linker is much lower than for mutant V66Az.


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)

Cyclic voltammogramsof the different mutants; lavender coloredline: mutant V66Az; rose colored line: mutant D314Az; lime green coloredline: background measurement with an unmodified working electrode.Scan rates were 0.05 V s–1; reference electrode:Ag/AgCl wire.
© Copyright Policy
Related In: Results  -  Collection

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

fig4: Cyclic voltammogramsof the different mutants; lavender coloredline: mutant V66Az; rose colored line: mutant D314Az; lime green coloredline: background measurement with an unmodified working electrode.Scan rates were 0.05 V s–1; reference electrode:Ag/AgCl wire.
Mentions: Figure 4 shows the cyclic voltammograms collected usinga mutant V66Az modified AFM tip (lavender colored line), a mutantD314Az modified tip (rose colored line), and a background measurementof the unmodified working electrode (lime green colored line) collectedat a scan rate of 0.05 V s–1. When studying the electrochemicalproperties of linker 1, based on eq 1, plotted Ip values against scan ratesdetermined a surface coverage of Γ = 2.3 × 10–10 mol·cm–2. However, surface coverage valuescalculated for the surface modified bacteria containing linker 1 bound to the surface of the electrode yielded values ofΓo = 8.0 × 10–11 mol·cm–2 and ΓR = 1.0 × 10–11 mol·cm–2 for mutant V66Az, oxidized and reducedforms, respectively, whereas the values of surface coverage for mutantD314Az were Γo = 2.7 × 10–11 mol·cm–2 and ΓR = 8.0 ×10–12 mol·cm–2, for the oxidizedand reduced forms. The much lower conversion values for the mutants, compared to thosemeasured for linker 1 by itself, suggest that there isan additional process that is involved, namely, the redox reactionof NADH. Taking into account the redox potential measured for mutantD314Az and the fact that it is identical to the redox potential oflinker 1, but with much lower reversibility, it is suggestedthat the degree of mediation of ET between NAD+ and thequinone containing linker is much lower than for mutant V66Az.

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