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

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.

SUBMITTER: Kuznetsova S

PROVIDER: S-EPMC2265120 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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

Kuznetsova Sofya S Zauner Gerhild G Aartsma Thijs J TJ Engelkamp Hans H Hatzakis Nikos N Rowan Alan E AE Nolte Roeland J M RJ Christianen Peter C M PC Canters Gerard W GW

Proceedings of the National Academy of Sciences of the United States of America 20080226 9

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. ...[more]

PMID: 18303118

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