EPR and Electron Nuclear Double Resonance (ENDOR) Studies Show Nitrite Binding to the Type 2 Copper Centers of the Dissimilatory Nitrite Reductase of Alcaligenes xylosoxidans (NCIMB 11015)

authored by: Barry D. Howes, Zelda H.L. Abraham, David J. Lowe, Thomas Brüser, Robert R. Eady, Barry E. Smith
Abstract: EPR and ¹H, ^14,15N ENDOR spectra are described for the type 1 and type 2 Cu(II) centers of dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans. The study was carried out on preparations of NiR containing both type 1 and type 2 Cu sites, and also on preparations of lower activity which contained essentially only type 1 Cu centers. This has enabled ENDOR studies of type 1 and type 2 sites to be carried out largely independently of each other, by appropriate choice of the excitation field. Spectra were recorded both in the absence and presence of nitrite, allowing a clear determination of which of the two types of Cu center constitutes the substrate binding site. The EPR results show large changes in the type 2 site g_ǁ (which decreases by 0.065) and ^CuA_ǁ (which increases by 2.0 mT) while the type 1 site EPR is not affected. In addition, both ¹H and ¹⁴N ENDOR of the type 2 Cu site undergo considerable changes on addition of nitrite whereas the type 1 Cu site ENDOR is unaffected. Our results clearly demonstrate that nitrite binds to the type 2 copper and that this process significantly perturbs the ligation of this copper by the protein histidine residues. No ¹⁵N ENDOR resonances were observed from ¹⁵N nitrite. The accessibility of the copper sites to solvent has been studied using ²H₂O. The results indicate that nitrite binds to the type 2 Cu by displacing a proton, probably on a water molecule bound to the copper atom.
External Organisation(s): University of Sussex
Type: Article
Journal: Biochemistry
Volume: 33
Pages: 3171-3177
No. of pages: 7
ISSN: 0006-2960
Publication date: 01.03.1994
Publication status: Published
Peer reviewed: Yes
ASJC Scopus subject areas: Biochemistry
Electronic version(s): https://doi.org/10.1021/bi00177a005 (Access: Unknown)

BibTeX

@article{e90753d3186c49e3ae901f93ffbb3079,
title = "EPR and Electron Nuclear Double Resonance (ENDOR) Studies Show Nitrite Binding to the Type 2 Copper Centers of the Dissimilatory Nitrite Reductase of Alcaligenes xylosoxidans (NCIMB 11015)",
abstract = "EPR and 1H, 14,15N ENDOR spectra are described for the type 1 and type 2 Cu(II) centers of dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans. The study was carried out on preparations of NiR containing both type 1 and type 2 Cu sites, and also on preparations of lower activity which contained essentially only type 1 Cu centers. This has enabled ENDOR studies of type 1 and type 2 sites to be carried out largely independently of each other, by appropriate choice of the excitation field. Spectra were recorded both in the absence and presence of nitrite, allowing a clear determination of which of the two types of Cu center constitutes the substrate binding site. The EPR results show large changes in the type 2 site gǁ (which decreases by 0.065) and CuAǁ (which increases by 2.0 mT) while the type 1 site EPR is not affected. In addition, both 1H and 14N ENDOR of the type 2 Cu site undergo considerable changes on addition of nitrite whereas the type 1 Cu site ENDOR is unaffected. Our results clearly demonstrate that nitrite binds to the type 2 copper and that this process significantly perturbs the ligation of this copper by the protein histidine residues. No 15N ENDOR resonances were observed from 15N nitrite. The accessibility of the copper sites to solvent has been studied using 2H2O. The results indicate that nitrite binds to the type 2 Cu by displacing a proton, probably on a water molecule bound to the copper atom.",
author = "Howes, {Barry D.} and Abraham, {Zelda H.L.} and Lowe, {David J.} and Thomas Br{\"u}ser and Eady, {Robert R.} and Smith, {Barry E.}",
note = "Copyright: Copyright 2017 Elsevier B.V., All rights reserved.",
year = "1994",
month = mar,
day = "1",
doi = "10.1021/bi00177a005",
language = "English",
volume = "33",
pages = "3171--3177",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "11",
}