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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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AFM retraction curves are shown for ADHII mutantsV66Az and D314Azduring cantilever attachment to a single bacterium. Adhesion forcesare visible for both mutants. Bare gold substrates are shown as acontrol.
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fig3: AFM retraction curves are shown for ADHII mutantsV66Az and D314Azduring cantilever attachment to a single bacterium. Adhesion forcesare visible for both mutants. Bare gold substrates are shown as acontrol.

Mentions: In order to ascertain gold–thiol bond formation betweenthe enzymes on the bacterial surface and the cantilevered tip, wemeasured the adhesion forces while engaging a single bacterium. Figure 3 shows examples of the measured adhesion using theforce mode for mutants D314Az and V66Az. As a control measurement,adhesion forces were also measured for a bare Au surface. In addition,only upon observing these adhesion forces could electrochemical activitybe detected on the surface of the AFM tip (serving as our workingelectrode). Combining all this evidence together with the actual sizeof our working electrode (ca. 100 μm2) indicatesthat the probe and system is capable of picking up a single bacteriumor a fragment. Control experiments conducted with surfaces modifiedwith WT nonmodified bacteria that were nonspecifically bound to thesurface as well as surface modified bacteria displaying WT-ADHII didnot yield any visible electrochemical signals. This occurred neitherwhen the probe engaged the bacteria on the surface nor when the probewas subsequently withdrawn.


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)

AFM retraction curves are shown for ADHII mutantsV66Az and D314Azduring cantilever attachment to a single bacterium. Adhesion forcesare visible for both mutants. Bare gold substrates are shown as acontrol.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: AFM retraction curves are shown for ADHII mutantsV66Az and D314Azduring cantilever attachment to a single bacterium. Adhesion forcesare visible for both mutants. Bare gold substrates are shown as acontrol.
Mentions: In order to ascertain gold–thiol bond formation betweenthe enzymes on the bacterial surface and the cantilevered tip, wemeasured the adhesion forces while engaging a single bacterium. Figure 3 shows examples of the measured adhesion using theforce mode for mutants D314Az and V66Az. As a control measurement,adhesion forces were also measured for a bare Au surface. In addition,only upon observing these adhesion forces could electrochemical activitybe detected on the surface of the AFM tip (serving as our workingelectrode). Combining all this evidence together with the actual sizeof our working electrode (ca. 100 μm2) indicatesthat the probe and system is capable of picking up a single bacteriumor a fragment. Control experiments conducted with surfaces modifiedwith WT nonmodified bacteria that were nonspecifically bound to thesurface as well as surface modified bacteria displaying WT-ADHII didnot yield any visible electrochemical signals. This occurred neitherwhen the probe engaged the bacteria on the surface nor when the probewas subsequently withdrawn.

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
Related in: MedlinePlus