title: Voltage-controlled fluorescence switching of a single redox protein.
abstract: Heterogeneous electron transfer (ET) of the redox protein, wild-type azurin (wt-Az) from Pseudomonas aeruginosa, was monitored at the single-molecule (SM) level by fluorescence resonance energy transfer (FRET), one electron at a time. Azurin molecules were labeled with an organic fluorophore (Cy5), and the FRET-coupling between Cy5 and the redox center (copper) was used to study ET to a semi-transparent, 10nm thin gold electrode in an optical configuration. By using a confocal microscope and a bipotentiostat for control of the electrode potential, the oxidation and reduction processes of individual Az-Cy5 molecules were monitored. In the oxidized state of the redox center of the azurin molecule, the fluorescence emission of the covalently attached Cy5 was largely quenched by FRET ('off'-state), whereas the emission was recovered upon reduction ('on'-state). The work presented here, shows directly controlled single redox switching events of an individual redox protein and its thermodynamic dispersion. We show that the distribution of midpoint potentials (E0) of individual azurin molecules peaks at 45.7±0.5 mV with a full width at half maximum of 15 mV vs saturated calomel electrode (SCE).
authors.affiliation: Leiden Institute of Physics, Huygens Laboratory, Leiden University, Leiden, The Netherlands. Electronic address: akkilic@physics.leidenuniv.nl.
authors.forename: Namik
authors.initials: N
authors.lastname: Akkilic
authors.affiliation: Leiden Institute of Physics, Huygens Laboratory, Leiden University, Leiden, The Netherlands.
authors.forename: Muhammad
authors.initials: M
authors.lastname: Kamran
authors.affiliation: Leiden Institute of Physics, Huygens Laboratory, Leiden University, Leiden, The Netherlands.
authors.forename: Razvan
authors.initials: R
authors.lastname: Stan
authors.affiliation: Leiden Institute of Physics, Huygens Laboratory, Leiden University, Leiden, The Netherlands.
authors.forename: Nusrat J M
authors.initials: NJ
authors.lastname: Sanghamitra
mesh_terms: D001400:Azurin / Q000737:chemistry*; D015374:Biosensing Techniques; D003300:Copper / Q000737:chemistry; D004579:Electron Transport; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D007700:Kinetics; D010084:Oxidation-Reduction; D011550:Pseudomonas aeruginosa / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D001400:Azurin; D003300:Copper
keywords: Cy5; Cyclic voltammetry; Fluorescence; Metalloprotein; Single molecule; Thermodynamics