Nitrite reduction by copper through ligand-mediated proton and electron transfer† †Electronic supplementary information (ESI) available: Synthetic and experimental procedures, spectral data and computational details. CCDC 1051416 and 1056984. For ESI and crystallographic data in CIF or other electronic format see DOI: 10.1039/c5sc00720h Click here for additional data file. Click here for additional data file.

Nitrite reductases (NiRs) are enzymes found in prokaryotic organisms which catalyze the one-electron (e ) reduction of nitrite to nitric oxide (NO). Copper nitrite reductases (CuNiRs) are homotrimeric enzymes with each monomer containing two copper centers: a T1 site for electron transfer, and a catalyticallyactive T2 site. Although X-ray crystallographic studies have provided structural snapshots of intermediates along the reduction pathway, the precise mechanism by which CuNiR catalyzes nitrite reduction has been disputed. In one proposed pathway, nitrite coordinates to the reduced T2 center, then following two proton (H) transfer events, water is released and a copper–nitrosyl species is generated (described as either Cu(I)– NO or Cu(II)–NO); Fig. 1A. In support of this mechanism, a crystal structure of CuNiR with NO bound to the reduced T2 site was reported with an unusual side-on binding mode of NO, which suggests that NO coordination to copper is at least possible under reducing conditions. However, an oxidized Cu(NO) unit (i.e. Fig. 1A) would be capable of nitrosylating nearby amino acid residues, and thus is unlikely to be formed under catalytic conditions. An alternative pathway for nitrite reduction catalyzed by CuNiR has been described wherein nitrite rst coordinates to the oxidized T2 center followed by H transfer from a nearby aspartic acid residue to protonate the coordinated nitrite. In this case, protonation of nitrite triggers e transfer from the T1 center to the T2 center, with release of NO to form a copper-hydroxide (Fig. 1B). This mechanism is consistent with isolated crystal structures of CuNiR with nitrite bound to the oxidized T2 center, steady-state kinetics and pulsed radiolysis experiments, and computational modeling. Although the intimate pathway of nitrite reduction may be