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The enzyme mechanism of nitrite reductase studied at single-molecule level.

Proceedings of the National Academy of Sciences of the United States of America (2008-02-28)
Sofya Kuznetsova, Gerhild Zauner, Thijs J Aartsma, Hans Engelkamp, Nikos Hatzakis, Alan E Rowan, Roeland J M Nolte, Peter C M Christianen, Gerard W Canters
ABSTRACT

A generic method is described for the fluorescence "readout" of the activity of single redox enzyme molecules based on Förster resonance energy transfer from a fluorescent label to the enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluorescence time traces can be connected to and agree with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type 1 to the type 2 center and back during turnover exhibit a distribution related to disorder in the catalytic site. The described approach opens the door to single-molecule mechanistic studies of a wide range of redox enzymes and the precise investigation of their internal workings.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Atto 655, BioReagent, suitable for fluorescence, ≥85% (HPLC)
Sigma-Aldrich
Atto 655 NHS ester, BioReagent, suitable for fluorescence
Sigma-Aldrich
Atto 655-Biotin, BioReagent, suitable for fluorescence, ≥95.0% (HPCE)
Sigma-Aldrich
Atto 655 amine, suitable for fluorescence
Sigma-Aldrich
Atto 655-maleimide, BioReagent, suitable for fluorescence, ≥90% (coupling rate)

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