Elucidating the Structures of the Low- and High-pH Mo(V) Species in Respiratory Nitrate Reductase: A Combined EPR,^14,15N HYSCORE, and DFT Study

Abstract

Respiratory nitrate reductases (Nars), members of the prokaryotic Mo/W-bis Pyranopterin Guanosine dinucleotide (Mo/W-bisPGD) enzyme superfamily, are key players in nitrate respiration, a major bioenergetic pathway widely used by microorganisms to cope with the absence of dioxygen. The two-electron reduction of nitrate to nitrite takes place at their active site, where the molybdenum ion cycles between Mo(VI) and Mo(IV) states via a Mo(V) intermediate. The active site shows two distinct pH-dependent Mo(V) electron paramagnetic resonance (EPR) signals whose structure and catalytic relevance have long been debated. In this study, we use EPR and HYSCORE techniques to probe their nuclear environment in Escherichia coli Nar (EcNar). By using samples prepared at different pH and through different enrichment strategies in Mo andN nuclei, we demonstrate that each of the two Mo(V) species is coupled to a single nitrogen nucleus with similar quadrupole characteristics. Structure-based density functional theory calculations allow us to propose a molecular model of the low-pH Mo(V) species consistent with EPR spectroscopic data. Our results show that the metal ion is coordinated by a monodentate aspartate ligand and permit the assignment of the coupled nitrogen nuclei to the Nδ of Asn52, a residue located ∼3.9 Å to the Mo atom in the crystal structures. This is confirmed by measurements on selectively N-Asn labeled EcNar. Further, we propose a Mo-O(H)···HN structure to account for the transfer of spin density onto the interacting nitrogen nucleus deduced from HYSCORE analysis. This work provides a foundation for monitoring the structure of the molybdenum active site in the presence of various substrates or inhibitors in Nars and other molybdenum enzymes.

Active site architecture reveals coordination sphere flexibility and specificity determinants in a group of closely related molybdoenzymes

“…Additionally, the Form 1 HiMtsZ spectrum observed here not only resembles previously determined EPR spectra of Dor/Tor-type enzymes, but also possesses a remarkable similarity to the lpH Mo V form of the NarGH Mo-containing nitrate reductase (43,44). This implies that both Form 1 HiMtsZ and lpH NarGH possess a similar 6-coordinate, distorted trigonal prismatic Mo V active site geometry (Figure 2E, Table S4), but with O Ser coordinated to the metal in HiMtsZ Form 1 instead of the O Asp donor that is bound to Mo in lpH NarGH.…”

“…Over the last three decades, a deep understanding of the molecular and catalytic properties of Mo enzymes has developed through the integration of structural biology, phylogenetic analysis, coordination chemistry, and spectroscopic studies (19,25,58). In the case of the Dor/Tor-type S-oxide/N-oxide reductases, while it is clear that they form a coherent phylogenetic group with very high structural similarity including at the Mo active site (Fig.…”

“…However, in addition to resembling published EPR signatures of the DorA S-oxide reductase (Table 3, Figure 2), the Form 1 & 2 HiMtsZ EPR spectra were also strikingly similar to those of the Mo-containing NarGH nitrate reductase, which has no significant sequence similarity to HiMtsZ or DorA (Figure S1) and possesses an aspartate ligand in place of serine. This suggests that the EPR spectroscopic properties of active sites with a MoS 4 O 2 first coordination sphere are primarily determined by the presence or absence of a hydroxide ligand, and the orientation of the strongly coupled proton relative to the 4d 1 redox orbital (43). These structural properties appear to be quite similar in HiMtsZ and NarGH, leading to very similar EPR spectra that are relatively unaffected by the identity of the amino acid ligand (O Asp /O Ser ) coordinated to the Mo center.…”

Identification of YdhV as the First Molybdoenzyme Binding a Bis-Mo-MPT Cofactor in Escherichia coli

“…A similarly rhombic 95,97 Mo hyperfine tensor likely reflects low coordination symmetry at the metal. As has been shown for the high-and low-pH Mo(V) EPRsignals of E. coli nitrate reductase, the dihedral angle between the oxo and aspartate ligands can modulate the Mo(V) g-values and the isotropic proton hyperfine interaction 66. The geometry of the two apical Mo(V) ligands may therefore also affect the EPR parameters of Mo(V) inYdhV.Based on EPR spin Hamiltonian parameters for bis-MGD enzymes in literature, two tentative Mo(V) coordination structures are conceivable for YdhV (Figure 10C,D).…”

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

“…EcNarGHI turnover induces a net translocation of protons across the membrane which contributes to maintaining the transmembrane proton gradient that drives, for instance, ATP synthesis. The NarG catalytic subunit holds the Mo-bisPyranopterin Guanosine Dinucleotide cofactor [11] and a FeS cluster [10,12,13]. The electron transfer subunit NarH harbors four FeS clusters [9,14].…”