H hyperfine spectroscopy and DFT modeling unveil the demethylmenasemiquinone binding mode to E. coli nitrate reductase A (NarGHI).

The quinol oxidation site Q in E. coli respiratory nitrate reductase A (EcNarGHI) reacts with the three isoprenoid quinones naturally synthesized by the bacterium, i.e. ubiquinones (UQ), menaquinones (MK) and demethylmenaquinones (DMK). The binding mode of the demethylmenasemiquinone (DMSK) intermediate to the EcNarGHI Q quinol oxidation site is analyzed in detail using H hyperfine (hf) spectroscopy in combination with HO/DO exchange experiments and DFT modeling, and compared to the menasemiquinone one bound to the Q site (MSK) previously studied by us. DMSK and MSK are shown to bind in a similar and strongly asymmetric manner through a short (~1.7 Å) H-bond. The origin of the specific hf pattern resolved on the DMSK field-swept EPR spectrum is unambiguously ascribed to slightly inequivalent contributions from two β-methylene protons of the isoprenoid side chain. DFT calculations show that their large isotropic hf coupling constants (A ~12 and 15 MHz) are consistent with both (i) a specific highly asymmetric binding mode of DMSK and (ii) a near in-plane orientation of its isoprenyl chain at Cβ relative to the aromatic ring, which differs by ~90° to that predicted for free or NarGHI-bound MSK. Our results provide new insights into how the conformation and the redox properties of different natural quinones are selectively fine-tuned by the protein environment at a single Q site. Such a fine-tuning most likely contributes to render NarGHI as an efficient and flexible respiratory enzyme to be used upon rapid variations of the Q-pool content.