TY  - JOUR
A1  - Grimaldi, Stéphane
A1  - Arias-Cartin, Rodrigo
A1  - Lanciano, Pascal
A1  - Lyubenova, Sevdalina
A1  - Endeward, Burkhard
A1  - Prisner, Thomas F.
A1  - Magalon, Axel
A1  - Guigliarelli, Bruno
T1  - Direct evidence for nitrogen ligation to the high stability semiquinone intermediate in Escherichia coli nitrate reductase A
T2  - Journal of biological chemistry
N2  - The membrane-bound heterotrimeric nitrate reductase A (NarGHI) catalyzes the oxidation of quinols in the cytoplasmic membrane of Escherichia coli and reduces nitrate to nitrite in the cytoplasm. The enzyme strongly stabilizes a menasemiquinone intermediate at a quinol oxidation site (Q(D)) located in the vicinity of the distal heme b(D). Here molecular details of the interaction between the semiquinone radical and the protein environment have been provided using advanced multifrequency pulsed EPR methods. (14)N and (15)N ESEEM and HYSCORE measurements carried out at X-band ( approximately 9.7 GHz) on the wild-type enzyme or the enzyme uniformly labeled with (15)N nuclei reveal an interaction between the semiquinone and a single nitrogen nucleus. The isotropic hyperfine coupling constant A(iso)((14)N) approximately 0.8 MHz shows that it occurs via an H-bond to one of the quinone carbonyl group. Using (14)N ESEEM and HYSCORE spectroscopies at a lower frequency (S-band, approximately 3.4 GHz), the (14)N nuclear quadrupolar parameters of the interacting nitrogen nucleus (kappa = 0.49, eta = 0.50) were determined and correspond to those of a histidine N(delta), assigned to the heme b(D) ligand His-66 residue. Moreover S-band (15)N ESEEM spectra enabled us to directly measure the anisotropic part of the nitrogen hyperfine interaction (T((15)N) = 0.16 MHz). A distance of approximately 2.2 Abetween the carbonyl oxygen and the nitrogen could then be calculated. Mechanistic implications of these results are discussed in the context of the peculiar properties of the menasemiquinone intermediate stabilized at the Q(D) site of NarGHI.
Y1  - 2021
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/76524
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-765247
SN  - 0021-9258
VL  - 285.2010
IS  - 1
SP  - 179
EP  - 187
PB  - American Society for Biochemistry and Molecular Biology Publications
CY  - Bethesda, Md
ER  -