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

Abstract

A generic method is described for the fluorescence ''readout'' of the activity of single redox enzyme molecules based on Fö rster resonance energy transfer from a fluorescent label to the enzyme cofactor. The method is applied to the study of copper-containing nitrite reductase from Alcaligenes faecalis S-6 immobilized on a glass surface. The parameters extracted from the single-molecule fluorescence time traces can be connected to and agree with the macroscopic ensemble averaged kinetic constants. The rates of the electron transfer from the type 1 to the type 2 center and back during turnover exhibit a distribution related to disorder in the catalytic site. The described approach opens the door to singlemolecule mechanistic studies of a wide range of redox enzymes and the precise investigation of their internal workings.electron transfer ͉ redox enzyme ͉ Fö rster transfer ͉ nitric oxide ͉ fluorescent label I n the past few years, single-enzyme studies have revealed numerous hidden aspects of enzyme behavior (1). The huge potential of these studies to unravel the intricate kinetics and precise workings of enzymes, which are often hidden within the ensemble properties, is somewhat restricted by current approaches. 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 (6-9). 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. Second, it is shown how the kinetics of the fluorescence time traces can be connected with the kinetic parameters that describe the ensemble behavior of the enzyme. Finally, the rate of the electron transfer between the types 1 and 2 Cu centers during turnover could be established. The observed distribution of rates is connected to the partial structural disorder in the catalytic site that has been observed in crystallographic studies. ResultsEnzyme Mechanism. Dissimilatory copper-containing nitrite reductase (NiR) from Alcaligenes faecalis S-6 converts nitrite into nitric oxide. The enzyme is a homotrimer, each monomer containing one type 1 and one type 2 copper site (Fig. 1). The type 1 copper accepts an electron from the physiological donor and transfers it to the type 2 copper, where nitrite is reduced to nitric oxide (NO). The midpoint potentials of the types 1 and 2 Cu centers are close, resulting in a redox equilibrium constant for the two sites that is close to 1 (12, 13). Regarding the enzyme mechanism, it has been stated (14, 15) that, after reduction of the type 1 Cu site, the electron is passed on to the type 2...

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

“…2 but at the moment is not required to explain the observed behavior. Molecule-to-molecule variability has begun to be quantified in immobilized NiR molecules (24,26).…”

“…This spectral change was recently employed to make a proximal dye molecule a fluorescent reporter of the T1 Cu oxidation state (24). The oxidized Cu significantly quenches the dye fluorescence via Förster resonant energy transfer (FRET) due to overlap between the dye's emission and the colored Cu's absorption, whereas the reduced colorless Cu leaves the dye unquenched and maximally emissive (24), Fig. 1.…”

“…1. Single green NiR enzymes (gNiR) (25) from Alcaligenes faecalis were immobilized and fluorescence intensity fluctuations were shown to be indicative of enzyme turnovers (24), although individual turnovers To whom correspondence may be addressed. E-mail: wmoerner@stanford.edu or canters@chem.leidenuniv.nl.…”

Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

“…In addition to changes in the redox potentials and thus in the driving force of the ET reaction, several structural changes in the redox centers have been reported as a result of substrate binding, which may also influence the inter-copper ET rate by changing the reorganization energy (16,25,30,31). These rearrangements include subtle changes in the Cys-His bridge linking T1-and T2Cu (32) and conformational transitions of the catalytically relevant active site residue Asp-92 (see below and Ref.…”

Microfabricated arrays of cylindrical wells facilitate single‐molecule enzymology of α‐chymotrypsin

“…Studies have shown that completing the experiment within turnover conditions may also ensure that photobleaching does not affect the accuracy of the experimental findings. 49 …”