title: 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.
authors.affiliation: Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
authors.forename: Sofya
authors.initials: S
authors.lastname: Kuznetsova
authors.forename: Gerhild
authors.initials: G
authors.lastname: Zauner
authors.forename: Thijs J
authors.initials: TJ
authors.lastname: Aartsma
authors.forename: Hans
authors.initials: H
authors.lastname: Engelkamp
authors.forename: Nikos
authors.initials: N
authors.lastname: Hatzakis
authors.forename: Alan E
authors.initials: AE
authors.lastname: Rowan
authors.forename: Roeland J M
authors.initials: RJ
authors.lastname: Nolte
authors.forename: Peter C M
authors.initials: PC
authors.lastname: Christianen
authors.forename: Gerard W
authors.initials: GW
authors.lastname: Canters
mesh_terms: D042481:Alcaligenes faecalis / Q000201:enzymology; D004579:Electron Transport; D004800:Enzymes, Immobilized; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D009572:Nitrite Reductases / Q000737:chemistry*
chemical_list: D004800:Enzymes, Immobilized; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing