Mechanism and Catalytic Site Atlas

Step 1. Nitrite substitutes water at the heme proximal coordination site.

Step 2. Ferredoxin donates one electron to the siroheme centre, via the iron-sulfur cluster, which is then shuttled to the nitrite ligand.

Step 3. A second Ferredoxin donates one electron to the siroheme centre which is again shuttled to the nitrite, followed by either deprotonation of a proximate basic residue or protonation by a solvent molecule.

Step 4. The reduced nitrate eliminates water. It is unclear whether residues or solvent molecules act as proton donors in this reaction. Conservation of Arg109, Arg223 and Lys224, and their close proximity to both the active site and a solvent channel suggests that these residues may be involved in providing protons for the reaction. Equally though, solvent molecules from the channel could act as proton donors [PMID:14717606]. EPR and kinetic studies have suggested the complex of ferrous iron and nitric oxide radical is formed after water has been lost [PMID:19226104].

Step 5. The iron centre donates a single electron to the nitric oxide radical, initiating protonation at the nitrogen.

Step 6. The active site residues are reprotonated for the next round of reduction. Crystallographic and circular diachrosim studies have shown the binding or lack of binding of ferredoxin to control solvent access to the active site [PMID:14717606]. It is postulated that when ferredoxon is not bound, the solvent channel opens, increasing the pKa of ionisable residues and reprotonating those thought to act as bases throughout the reaction.

Step 7. Another molecule of reduced ferredoxin donates a single electron through the cluster and heme cofactors to the nitrogen-oxygen ligand.

Step 8. Another molecule of reduced ferredoxin donates a single electron through the cluster and heme cofactors, this time to the radical nitrogen-oxygen ligand, forming hydroxylamine. While very little information is known about the reaction intermediates of nitrate reductase, kinetic studies have shown the presence of hydroxylamine, the intermediate formed in this step, to drive the reduction of ascorbate (a non-physiological reduction equivalent to ferredoxin), change the spectroscopic properties of the complex, and form ammonia suggesting it is a reaction intermediate [PMID:20039132].

Step 9. The active site residues are reprotonated for the next round of reduction

Step 10. Reduced ferredoxin donates a single electron through the cluster and heme cofactors initiating the elimination of water.

Step 11. Reduced ferredoxin donates a single electron through the cluster and heme cofactors to the axial nitrogen ligand, initiating protonation to form ammonia.

Step 12. The ammonia ligand is protonated and substituted by a water molecule. The departing ammonia could abstract a proton from either a solvent molecule located in the solvent channel, or from a protonated residue within the active site.

Step 13. The ionisable residues surrounding the active site are reprotonated in the absence of ferredoxin binding. As inferred from structural and spectroscopic studies, the ionisable residues proxmiate to the active site and solvent channel are reprotonated for the next round of catalysis [PMID:19226104].

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