Fig. 1.

Sequence of events during the turnover of NiR labeled on the N terminus with ATTO 655. (Left) In the resting enzyme, both types 1 (blue) and Cu (red) are oxidized, and the ATTO 655 fluorescence is low because of FRET to the oxidized type 1 Cu. (Center) Upon reduction of type 1 Cu by an external electron donor, the dye fluorescence goes up, because FRET from ATTO 655 to the reduced type 1 Cu (gray) is not possible. (Right) An electron is transferred from types 1 to 2 Cu, which becomes reduced (gray). The label fluorescence is quenched by the oxidized type 1 Cu. Nitrite binds to the reduced type 2 Cu, is converted into nitric oxide, and dissociates from the enzyme.