Insights into periplasmic nitrate reductase function under single turnover

Abstract

Nitrate reductases play pivotal roles in nitrogen metabolism by leveraging the molybdopterin cofactor to facilitate the reduction of nitrate to nitrite. Periplasmic nitrate reductases (NapA) utilize nitrate as a terminal electron acceptor when oxygen is limiting, helping to drive anaerobic metabolism in bacteria. Despite extensive research into NapA homologs, open questions about the mechanism remain especially at the molecular level. More broadly, little is understood of how the molybdopterin cofactor is tuned for catalysis in these enzymes enabling broad substrate scope and reactivity observed in molybdenum-containing enzymes. Here, we have prepared NapA from Campylobacter jejuni under single turnover conditions to generate a singly reduced enzyme that can be further examined by electron paramagnetic resonance (EPR) spectroscopy. Our results provide new context into the known spectra and related structures of NapA and related enzymes. These insights open new avenues for understanding nitrate reductase mechanisms, molybdenum coordination dynamics, and the role of pyranopterin ligands in catalysis.

Keywords: Campylobacter jejuni; Electron paramagnetic resonance; Molybdenum; Molybdopterin; Nitrate reductase.

Figure 1:. Mechanism and active site structure of periplasmic nitrate reductases.

(A) The reaction mechanism proposed for NapA resembles many oxygen atom transfer reactions catalyzed by molybdenum enzymes. As with many molybdenum- and tungsten-containing enzymes, the metal cycles through single electron reduction/oxidation and formal two-electron oxidation/reduction events during catalysis. The reducing equivalents in NapA are supplied to Mo sequentially by a nearby ironsulfur cluster near the proximal pyranopterin. The EPR-active species, highlighted in green, is generated via substrate reduction and internal electron transfer. The cofactor (B) resembles that found in NapA from Desulfovibrio desulfuricans (PDB ID: 2jip), where Mo is coordinated by two inequivalent pyranopterins, present as the pyranopterin guanosine dinucleotide (the dinucleotide moiety is shown as R), a cysteine. Lewis structures of the distal and proximal pyranopterins are provided for clarity.

Figure 2:

EPR Spectra of NapA. EPR spectra (Black) and simulations (Red) of the wildtype NapA reduced with methyl viologen (MV) and partially oxidized with nitrate. When immediately frozen after addition of nitrate we observe a single species (A), termed ‘Species 1’, When A is buffer exchanged, a complex spectrum comprised of three species is observed (B). The two major components are a newly identified ‘Species 2’ (x) and some remaining ‘Species 1’ (y). The simulation also includes an organic radical (z) that corresponds to 1-2% of the sample. EPR spectra were recorded at 150K, 9.4 GHz, 2 mW power, 3 G modulation amplitude.

Figure 3.

Structure Models of NapA with and without nitrite near the molybdenum cofactor but distant from the site of catalysis. Nitrite modeled in the crystal structure (PDB: 2jiq, left), circled in red, is below the plane of the pyranopterins in a location occupied by two water molecules in other structure models (PDB:2jip, right). We note additional movements of Arg617 in the structure that hydrogen bonds to N-5 of one pyranopterin (blue circle).