Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase

  • Posted on: 10 July 2014
  • By: admin
TitleRedox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase
Publication TypeJournal Article
Year of Publication2011
AuthorsGoldsmith R.H, Tabares L.C, Kostrz D., Dennison C., Aartsma T.J, Canters G.W, Moerner W.E
JournalProceedings of the National Academy of Sciences of the United States of America
Volume108
Pagination17269-17274
Date PublishedOct 18
ISBN Number0027-8424
Accession NumberISI:000295975300016
Keywordsabel trap, achromobacter cycloclastes, alcaligenes-xylosoxidans, blue, brownian-motion, catalysis, dynamics, electron transfer, enzymology, fluorescence, intramolecular electron-transfer, michaelis-menten equation, ordered mechanism, type-2 cu
Abstract

Single-molecule measurements are a valuable tool for revealing details of enzyme mechanisms by enabling observation of unsynchronized behavior. However, this approach often requires immobilizing the enzyme on a substrate, a process which may alter enzyme behavior. We apply a microfluidic trapping device to allow, for the first time, prolonged solution-phase measurement of single enzymes in solution. Individual redox events are observed for single molecules of a blue nitrite reductase and are used to extract the microscopic kinetic parameters of the proposed catalytic cycle. Changes in parameters as a function of substrate concentration are consistent with a random sequential substrate binding mechanism.

Alternate JournalP Natl Acad Sci USA