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Electrochemical Single-Molecule AFM of the Redox Metalloenzyme Copper Nitrite Reductase in Action

Authors

  • Dr. Xian Hao,

    1. Department of Chemistry, Building 207, Technical University of Denmark, DK-2800 Kongens Lyngby (Denmark)
    2. Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China)
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  • Prof. Dr. Jingdong Zhang,

    1. Department of Chemistry, Building 207, Technical University of Denmark, DK-2800 Kongens Lyngby (Denmark)
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  • Prof. Dr. Hans E. M. Christensen,

    1. Department of Chemistry, Building 207, Technical University of Denmark, DK-2800 Kongens Lyngby (Denmark)
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  • Prof. Dr. Hongda Wang,

    1. Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022 (China)
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  • Prof. Dr. Jens Ulstrup

    Corresponding author
    1. Department of Chemistry, Building 207, Technical University of Denmark, DK-2800 Kongens Lyngby (Denmark)
    • Department of Chemistry, Building 207, Technical University of Denmark, DK-2800 Kongens Lyngby (Denmark)
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Abstract

We studied the electrochemical behavior of the redox metalloenzyme copper nitrite reductase (CNiR, Achromobacter xylosoxidans) immobilized on a Au(111)-electrode surface modified by a self-assembled cysteamine molecular monolayer (SAM) using a combination of cyclic voltammetry and electrochemically-controlled atomic force microscopy (in situ AFM). The enzyme showed no voltammetric signals in the absence of nitrite substrate, whereas a strong reductive electrocatalytic signal appeared in the presence of nitrite. Such a pattern is common in protein film and monolayer voltammetry and points to conformational changes in the enzyme upon substrate binding. Binding thus either improves the enzyme/electrode contact, or opens intramolecular electron-transfer channels between the redox center for electron inlet (a type I copper center) and the catalytic site for nitrite reduction (a type II copper center). The in situ AFM data are at the level of the single CuNiR enzyme molecule. The voltammetric patterns were paralleled by a clear increase (swelling) of the molecular height when the electrochemical potential traversed the region from resting to the electrocatalytically active redox enzyme function in the presence of nitrite. No change in size was observed in the absence of nitrite over the same potential range. The enzyme size variation is suggested to offer clues to the broadly observed substrate triggering in metalloenzyme monolayer voltammetry.

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