title: Redox cycling and kinetic analysis of single molecules of solution-phase nitrite reductase.
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.
authors.affiliation: Department of Chemistry, Stanford University, Stanford, CA 94305, USA.
authors.forename: Randall H
authors.initials: RH
authors.lastname: Goldsmith
authors.forename: Leandro C
authors.initials: LC
authors.lastname: Tabares
authors.forename: Dorota
authors.initials: D
authors.lastname: Kostrz
authors.forename: Christopher
authors.initials: C
authors.lastname: Dennison
authors.forename: Thijs J
authors.initials: TJ
authors.lastname: Aartsma
authors.forename: G W
authors.initials: GW
authors.lastname: Canters
authors.forename: W E
authors.initials: WE
authors.lastname: Moerner
mesh_terms: D000421:Alcaligenes / Q000201:enzymology / Q000235:genetics; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D003300:Copper / Q000737:chemistry; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D046210:Microfluidic Analytical Techniques; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009572:Nitrite Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D020836:Protein Structure, Quaternary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012996:Solutions
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D012996:Solutions; D003300:Copper; D009572:Nitrite Reductases