Probing the Menasemiquinone Binding Mode to Nitrate Reductase A by Selective ²H and ¹⁵N Labeling, HYSCORE Spectroscopy, and DFT Modeling

Abstract

In vivo specific isotope labeling at the residue or substituent level is used to probe menasemiquinone (MSK) binding to the quinol oxidation site of respiratory nitrate reductase A (NarGHI) from E. coli. 15N selective labeling of His15Nδ or Lys15Nζ in combination with hyperfine sublevel correlation (HYSCORE) spectroscopy unambiguously identified His15Nδ as the direct hydrogen‐bond donor to the radical. In contrast, an essentially anisotropic coupling to Lys15Nζ consistent with a through‐space magnetic interaction was resolved. This suggests that MSK does not form a hydrogen bond with the side chain of the nearby Lys86 residue. In addition, selective 2H labeling of the menaquinone methyl ring substituent allows unambiguous characterization of the 2H—and hence of the 1H—methyl isotropic hyperfine coupling by 2H HYSCORE. DFT calculations show that a simple molecular model consisting of an imidazole Nδ atom in a hydrogen‐bond interaction with a MSK radical anion satisfactorily accounts for the available spectroscopic data. These results support our previously proposed one‐sided binding model for MSK to NarGHI through a single short hydrogen bond to the Nδ of His66, one of the distal heme axial ligands. This work establishes the basis for future investigations aimed at determining the functional relevance of this peculiar binding mode.

Density functional theory and ab initio studies on hyperfine coupling constants of phosphinyl radicals

“…Among them are electronic (UV/Vis), vibrational (infrared and Raman), nuclear magnetic resonance (NMR), and electron paramagnetic resonance (EPR) spectroscopy. The latter—also called electron spin resonance (ESR) spectroscopy—has proved to be indispensable in studies of radical species, which attract the attention of researchers due to their important roles in physics, chemistry, biochemistry, environmental, and material science …”

“…However, modern computational techniques can predict spectral data with an accuracy sufficient for a meaningful comparison with their experimental counterparts, and thus in such difficult situations they can support or verify the conclusions of experiments or link spectral features to the structural properties . Computational methods have proven especially successful in the interpretation of EPR data acquired experimentally for a broad representation of radicals based on the first‐row elements . However, to date, significantly less attention has been paid to the applications of theoretical methods to the properties of radicals with an unpaired electron significantly located on the heavier atoms, even though such systems draw the significant attention of researchers .…”

1,2H hyperfine spectroscopy and DFT modeling unveil the demethylmenasemiquinone binding mode to E. coli nitrate reductase A (NarGHI)

“…Intriguingly, EPR-monitored redox titrations indicated that, in contrast to MSK and USQ [22,27], the two-electron midpoint potential of EcNarGHI-bound DMK is decreased by at least 30 mV with respect to that of the unbound species [25]. This shift likely results from a redox-dependent differential binding of DMK at the Q D site corresponding to a ~10 fold tighter binding of DMK than DMKH 2 to this site.…”

“…In addition, in contrast to USQ D and MSK D , the DMSK D EPR signal exhibits a resolved hf structure from one or several nearby non exchangeable protons [19] whose origin was unclear. Its linewidth has been shown to decrease upon H 2 O/ 2 H 2 O exchange, revealing the existence of at least one exchangeable proton weakly coupled to DMSK D [25].…”

“…Based on multifrequency 14,15 N ESEEM/HYSCORE measurements [23], site-directed mutagenesis studies [22,24] and selective 15 N labeling [25], this interacting nucleus has been ascribed to the N  imidazole nitrogen of the heme b D axial ligand His66. Combining the use of H 2 O/ 2 H 2 O exchange experiments, selective 2 H labeling of the ring methyl protons and 1,2 H hyperfine spectroscopies, we have shown that MSK D is stabilized thanks to a short in-plane H-bond to the quinone oxygen O1, leading to a highly asymmetric distribution of the electron spin density over the semiquinone ring in comparison with the unbound species [25,26].…”