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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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(A)Cyclic voltammograms conducted under different scan rates formutant V66Az. (B) Cyclic voltammograms conducted under different scanrates for mutant D314Az. The range was limited to emphasize the peaks.The full scale is shown in Figure S2A,B, SupportingInformation.
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fig5: (A)Cyclic voltammograms conducted under different scan rates formutant V66Az. (B) Cyclic voltammograms conducted under different scanrates for mutant D314Az. The range was limited to emphasize the peaks.The full scale is shown in Figure S2A,B, SupportingInformation.

Mentions: We have usedLaviron’s analytical approach for cases of peak to peak separationof ΔEp > 200 mV/n (n being the number of electrons)25 to calculatethe transfer coefficient α and the apparent rate constant kapp for mutants V66Az and D314Az. We did notconduct calculations for mutant P182Az since it has exhibited similarpeak potentials as mutant V66Az, probably due to similar distancesfrom the NAD+ binding pocket. Figure 5A shows the voltammograms collected upon picking up a bacterium thatdisplayed ADHII mutant V66Az on its surface. Due to very low peakcurrents compared to catalytic currents present in the voltammograms,we are not showing the full range of potentials that were scannedin each experiment, only the region in which the peaks have appeared(the full scale voltammograms are shown in Figure S2A, Supporting Information). For mutant V66Az, theformal potential, E0′ was calculatedto be −250 mV vs Ag/AgCl. This relatively high potential isan indication that indeed the electrons are being transferred fromNADH through the quinone and not just from the quinone that is directlybound to the surface. The middle point potential that was measuredfor the quinone used in this study is −350 mV vs Ag/AgCl (FigureS2B, Supporting Information). Transfercoefficients α and 1 – α were calculated to be0.4 and 0.6, respectively, whereas kapp, the electron transfer rate constant, varied in the different measurementsbetween 5.6 and 7.2 s–1. These values are in goodagreement with values reported in the literature for electrodes modifiedwith quinone derivatives to mediate NADH enzymatic oxidation, wherethe enzymes were randomly oriented relative to the electrode.26 These values are significantly higher than valuesreported for ADH/toluidine blue O/nafion electrodes modified nonspecifically,at a value of 0.12 s–1.19


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)

(A)Cyclic voltammograms conducted under different scan rates formutant V66Az. (B) Cyclic voltammograms conducted under different scanrates for mutant D314Az. The range was limited to emphasize the peaks.The full scale is shown in Figure S2A,B, SupportingInformation.
© Copyright Policy
Related In: Results  -  Collection

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

fig5: (A)Cyclic voltammograms conducted under different scan rates formutant V66Az. (B) Cyclic voltammograms conducted under different scanrates for mutant D314Az. The range was limited to emphasize the peaks.The full scale is shown in Figure S2A,B, SupportingInformation.
Mentions: We have usedLaviron’s analytical approach for cases of peak to peak separationof ΔEp > 200 mV/n (n being the number of electrons)25 to calculatethe transfer coefficient α and the apparent rate constant kapp for mutants V66Az and D314Az. We did notconduct calculations for mutant P182Az since it has exhibited similarpeak potentials as mutant V66Az, probably due to similar distancesfrom the NAD+ binding pocket. Figure 5A shows the voltammograms collected upon picking up a bacterium thatdisplayed ADHII mutant V66Az on its surface. Due to very low peakcurrents compared to catalytic currents present in the voltammograms,we are not showing the full range of potentials that were scannedin each experiment, only the region in which the peaks have appeared(the full scale voltammograms are shown in Figure S2A, Supporting Information). For mutant V66Az, theformal potential, E0′ was calculatedto be −250 mV vs Ag/AgCl. This relatively high potential isan indication that indeed the electrons are being transferred fromNADH through the quinone and not just from the quinone that is directlybound to the surface. The middle point potential that was measuredfor the quinone used in this study is −350 mV vs Ag/AgCl (FigureS2B, Supporting Information). Transfercoefficients α and 1 – α were calculated to be0.4 and 0.6, respectively, whereas kapp, the electron transfer rate constant, varied in the different measurementsbetween 5.6 and 7.2 s–1. These values are in goodagreement with values reported in the literature for electrodes modifiedwith quinone derivatives to mediate NADH enzymatic oxidation, wherethe enzymes were randomly oriented relative to the electrode.26 These values are significantly higher than valuesreported for ADH/toluidine blue O/nafion electrodes modified nonspecifically,at a value of 0.12 s–1.19

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