Sensitive detection of the redox state of copper proteins using fluorescence

Abstract

The blue copper protein azurin from Pseudomonas aeruginosa has been covalently labelled with the fluorescing dye Cy5. The optical spectrum of the azurin changes markedly with its redox state. These changes are reflected in the fluorescence intensity of the dye through fluorescence resonance energy transfer (FRET). This provides a sensitive way to monitor biological redox events. The method shown to work in the nanomolar range of protein concentrations, can be easily extended into the sub-nanomolar regime and holds promise for single-molecule detection.

The enzyme mechanism of nitrite reductase studied at single-molecule level

“…The L93C and L93C/H145A variants of nitrite reductase from A. faecalis S-6 were expressed and purified as described (17,38). Both L93C and L93C/H145A NiR mutants were labeled with ATTO 655 succinimidyl ester (ATTO-TEC Biolabeling and Ultraanalytics) on the N terminus using a 5ϫ molar excess of dye over the protein and purified by using Centrispin-10 size-exclusion chromatography spin columns with a 5-kDa cutoff (Princeton Separations) (10,11). The degree of labeling has been quantified taking 665 ϭ 125 mM Ϫ1 ⅐cm Ϫ1 for ATTO 655 and 280 ϭ 46 mM Ϫ1 ⅐cm Ϫ1 per monomer for both nitrite reductase mutants (17).…”

“…The majority of existing single-molecule enzymatic assays are based on fluorescence and have been limited to the flavoenzymes, which contain a fluorescent cofactor (2-5), or to enzymes for which a suitable fluorogenic substrate could be designed (6)(7)(8)(9). Recently, it was shown how redox enzyme activity in the bulk can be studied by Förster resonance energy transfer (FRET) from an attached fluorescent label to the enzyme cofactor (10,11). Here, we report, first, how this technique can be successfully applied to study the enzymatic turnover of single surface-confined copper-containing nitrite reductase (NiR) molecules labeled with ATTO 655 using scanning confocal fluorescence microscopy.…”

Fluorescence Lifetime Analysis of Nitrite Reductase from Alcaligenes xylosoxidans at the Single‐Molecule Level Reveals the Enzyme Mechanism

“…Quenching is the result of Fçrster resonant energy transfer (FRET). [19][20][21][22] Thus, fluctuations in the redox state of the T1 copper site during turnover are reflected in fluorescence intensity variations. [19][20][21][22][23] The application of this scheme to the study of the electrochemical behavior of electron-transfer proteins has recently been reported.…”

“…[19][20][21][22] Thus, fluctuations in the redox state of the T1 copper site during turnover are reflected in fluorescence intensity variations. [19][20][21][22][23] The application of this scheme to the study of the electrochemical behavior of electron-transfer proteins has recently been reported. [24] In earlier studies on the green NiR (gNiR; Figure S4 in the Supporting Information) from Alcaligenes faecalis-S6, the intensity distributions in fluorescence time traces of single molecules during turnover were analyzed by means of an autocorrelation analysis.…”

The Oxidation State of a Protein Observed Molecule‐by‐Molecule

“…Previously, we demonstrated a novel fluorescence-based detection scheme for this transition. [6] The methodology is based on the redox-state-dependent change in absorption of the nonfluorescent copper center, which quenches, by fluorescence resonance energy transfer (FRET), the fluorescence of a small organic fluorophore conjugated to the protein. The change in absorption of the copper center modulates the spectral overlap and thus results in a change in Fçrster radius and energy transfer efficiency.…”