TY  - JOUR
A1  - Grimaldi, Stéphane
A1  - Arias-Cartin, Rodrigo
A1  - Lanciano, Pascal
A1  - Lyubenova, Sevdalina
A1  - Szenes, Rodolphe
A1  - Endeward, Burkhard
A1  - Prisner, Thomas F.
A1  - Guigliarelli, Bruno
A1  - Magalon, Axel
T1  - Determination of the proton environment of high stability Menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR
T2  - Journal of biological chemistry
N2  - Escherichia coli nitrate reductase A (NarGHI) is a membrane-bound enzyme that couples quinol oxidation at a periplasmically oriented Q-site (Q(D)) to proton release into the periplasm during anaerobic respiration. To elucidate the molecular mechanism underlying such a coupling, endogenous menasemiquinone-8 intermediates stabilized at the Q(D) site (MSQ(D)) of NarGHI have been studied by high-resolution pulsed EPR methods in combination with (1)H2O/2H2O exchange experiments. One of the two non-exchangeable proton hyperfine couplings resolved in hyperfine sublevel correlation (HYSCORE) spectra of the radical displays characteristics typical from quinone methyl protons. However, its unusually small isotropic value reflects a singularly low spin density on the quinone carbon α carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ(D) binding mode and consistent with single-sided hydrogen bonding to the quinone oxygen O1. Furthermore, a single exchangeable proton hyperfine coupling is resolved, both by comparing the HYSCORE spectra of the radical in 1H2O and 2H2O samples and by selective detection of the exchanged deuterons using Q-band 2H Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i.e. a large anisotropic hyperfine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short ∼1.6 Å in-plane hydrogen bond between the quinone O1 oxygen and the Nδ of the His-66 residue, an axial ligand of the distal heme b(D). Structural and mechanistic implications of these results for the electron-coupled proton translocation mechanism at the Q(D) site are discussed, in light of the unusually high thermodynamic stability of MSQ(D).
KW  - Bioenergetics
KW  - Electron Paramagnetic Resonance (EPR)
KW  - Electron Transfer
KW  - Metalloenzymes
KW  - Quinones
Y1  - 2021
UR  - http://publikationen.ub.uni-frankfurt.de/frontdoor/index/index/docId/76670
UR  - https://nbn-resolving.org/urn:nbn:de:hebis:30:3-766700
SN  - 0021-9258
VL  - 287.2012
IS  - 7
SP  - 4662
EP  - 4670
PB  - American Society for Biochemistry and Molecular Biology Publications
CY  - Bethesda, Md
ER  -