Determination of the proton environment of high stability menasemiquinone intermediate in Escherichia coli nitrate reductase A by pulsed EPR

Abstract

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 1H 2O/ 2H 2O 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 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 1H 2O and 2H 2O 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 inplane 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 Dsite are discussed, in light of the unusually high thermodynamic stability of MSQ D. © 2012 by The American Society for Biochemistry and Molecular Biology, Inc.