Letter to the Editor:1H,13C and15N resonance assignment of Cu(I)-pseudoazurin from Alcaligenes faecalis S-6

Impagliazzo, Antonietta; Ubbink, Marcellus

doi:10.1023/b:jnmr.0000034341.96429.c1

Cited by 9 publications

(28 citation statements)

References 7 publications

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“…Periods of high fluorescence alternate on a time scale of seconds with dark periods in which only the background signal is observed; we ascribe this to dye blinking (21)(22)(23)(24). The steady nature of the observed fluorescence during the ''on'' periods is consistent with crystallographic and NMR evidence, indicating absence of largescale conformational fluctuations (18,25). ¶ In passing, we note that the one-step bleaching (data not shown) and blinking observed under nonturnover conditions confirmed we were monitoring single molecules.…”

Section: Resultssupporting

confidence: 72%

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

Кузнецова

Zauner

Aartsma

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultssupporting

confidence: 72%

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

Кузнецова

Zauner

Aartsma

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

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“…Uniformly 15 N-labelled PAZ was isolated and purified as described. [20] To replace copper with zinc in PAZ, a solution of KCN (200 mm, 0.5 mL) in Tris-HCl (0.5 m, pH 7.0) was added to PAZ Cu II (1 mm, 0.5 mL) in Tris-HCl (0.1 m, pH 7.0). As soon as the solution had bleached, loosing the characteristic blue colour of PAZ Cu II , the sample was loaded onto a G25 column equilibrated with Tris-HCl (0.1 m, pH 7.0) containing ZnCl 2 (1 mm).…”

Section: Methodsmentioning

confidence: 99%

“…All spectra were processed in AZARA (http://www.bio.cam.ac.uk/ azara/) and analysed with the assignment programme ANSIG for Windows. [23,24] Assignments for 15 N-PAZ Cu I [20] and 15 N-PAZ Zn II [10] were taken from the literature.…”

Section: àCumentioning

confidence: 99%

Pseudoazurin–Nitrite Reductase Interactions

2005

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The nitrite reductase-binding site on pseudoazurin has been determined by using NMR chemical-shift perturbations. It comprises residues in the hydrophobic patch surrounding the exposed copper ligand His81 as well as several positively charged residues. The binding site is similar for both redox states of pseudoazurin, despite differences in the binding mode. The results suggest that pseudoazurin binds in a well-defined orientation. Docking simulations provide a putative structure of the complex with a binding site on nitrite reductase that has several hydrophobic and polar residues as well as a ridge of negatively charged side chains and a copper-to-copper distance of 14 A.

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“…42 The assignments of the 15 N-Zn-Paz HSQC spectra were taken from the literature. 18,43 Chemical shift perturbation analysis…”

Section: Nmr Spectroscopymentioning

confidence: 99%