Determination of the Proton Environment of High Stability Menasemiquinone Intermediate in Escherichia coli Nitrate Reductase A by Pulsed EPR

作者：Grimaldi Stephane^*; Arias Cartin Rodrigo; Lanciano Pascal; Lyubenova Sevdalina; Szenes Rodolphe; Endeward Burkhard; Prisner Thomas F; Guigliarelli Bruno; Magalon Axel

来源：Journal of Biological Chemistry, 2012, 287(7): 4662-4670.

DOI：10.1074/jbc.M111.325100

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 (H2O)-H-1/(H2O)-H-2 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 alpha carrying the methyl group, which is ascribed to a strong asymmetry of the MSQ(D) binding mode and consistent with singlesided 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 (H2O)-H-1 and (H2O)-H-2 samples and by selective detection of the exchanged deuterons using Q-band H-2 Mims electron nuclear double resonance (ENDOR) spectroscopy. Spectral analysis reveals its peculiar characteristics, i. e. a large anisotropic hyper-fine coupling together with an almost zero isotropic contribution. It is assigned to a proton involved in a short similar to 1.6 angstrom inplane hydrogen bond between the quinone O1 oxygen and the N delta 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).

• 出版日期2012-2-10