The enzyme mechanism of nitrite reductase studied at single-molecule level.
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.
Kuznetsova
Sofya
S
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, P.O. Box 9502, 2300 RA, Leiden, The Netherlands.
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
D042481:Alcaligenes faecalis / Q000201:enzymology; D004579:Electron Transport; D004800:Enzymes, Immobilized; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D009572:Nitrite Reductases / Q000737:chemistry*
D004800:Enzymes, Immobilized; D009572:Nitrite Reductases; C052695:nitrite reductase, copper-containing