With limited reductant and nitrite under anaerobic conditions, copper-containing nitrite reductase (NiR) of Rhodobacter sphaeroides yielded endogenous NO and the Cu(I)NO derivative of NiR. (14)N- and (15)N-nitrite substrates gave rise to characteristic (14)NO and (15)NO EPR hyperfine features indicating NO involvement, and enrichment of NiR with (63)Cu isotope caused an EPR line shape change showing copper involvement. A markedly similar Cu(I)NONiR complex was made by anaerobically adding a little endogenous NO gas to reduced protein and immediately freezing. The Cu(I)NONiR signal accounted for 60-90% of the integrated EPR intensity formerly associated with the Type 2 catalytic copper. Analysis of NO and Cu hyperfine couplings and comparison to couplings of inorganic Cu(I)NO model systems indicated approximately 50% spin on the N of NO and approximately 17% spin on Cu. ENDOR revealed weak nitrogen hyperfine coupling to one or more likely histidine ligands of copper. Although previous crystallography of the conservative I289V mutant had shown no structural change beyond the 289 position, this mutation, which eliminates the Cdelta1 methyl of I289, caused the Cu(I)NONiR EPR spectrum to change and proton ENDOR features to be significantly altered. The proton hyperfine coupling that was significantly altered was consistent with a dipolar interaction between the Cdelta1 protons of I289 and electron spin on the NO, where the NO would be located 3.0-3.7 A from these protons. Such a distance positions the NO of Cu(I)NO as an axial ligand to Type 2 Cu(I).

Copper nitrite reductase (NiR) is a homotrimeric enzyme, containing a T1 copper site, which transfers electrons to the T2 catalytic site, where nitrite is reduced by one electron to nitric oxide (NO 2 − + 2H + + e − → NO + H 2 O). 1-3 Recently a side-on bound copper nitrosyl complex of NiR has been crystallized by reacting the reduced enzyme with excess NO. 4 Based on the EPR spectrum of the species generated by the reaction of reduced NiR with saturated NO in solution, it has been assigned as an η 2 -NO − Cu 2+ species. However, spectroscopic data on several Cu-nitrosyl model complexes 5 and an NO adduct formed by reacting reduced NiR with nitrite 6 and DFT calculations on an η 2 -NOCu species 7 describe these complexes as Cu + NO • . In this study, we define the species generated in solution by reacting reduced NiR with NO, using EPR, MCD and ENDOR spectroscopy and correlate these with that observed by crystallography.Reduced NiR reacted with saturated NO solution generates the EPR spectrum in Figure 1A red, which is equivalent to the spectrum reported by Tocheva et al. 4 This species has an EPR signal characteristic of an oxidized T2 Cu (g || = 2.30 and A || = 117 G). The MCD spectrum (Fig. 1B, red) has low energy d-d bands, also indicative of a T2 cupric complex. Both EPR and MCD spectra show a negligible T1 contribution indicating that this site is reduced (Fig. S1).Nitrite binds to the T2 site of the resting WT enzyme, 8,9 but the EPR and, in particular, the MCD features of the nitrite bound T2 site are obscured by the dominant T1 Cu signals. However under these conditions (nitrite bound), the T1 Cu could be selectively reduced with acsorbate. The EPR spectrum of the nitrite bound T2 species (Fig. 1A, blue) is identical to that produced above by reacting reduced NiR with excess NO. The MCD spectrum (Fig. 1B, blue) of the T1 reduced T2-nitrite bound form is also identical to that generated by the reduced enzyme with NO. The T2 EPR spectrum and the MCD d-d bands of the resting WT enzyme are very sensitive to nitrite binding (Fig. S1C, D). Thus the EPR and MCD data strongly indicate that the species generated by the reaction of excess NO with reduced NiR has T1 reduced and T2 oxidized with nitrite bound.Further N 14 and H 1 ENDOR data for this species generated by the reaction of reduced NiR with an NO solution (Fig. 1C, D, red) and those for a form with T1+T2 oxidized and nitrite bound (collected at g=2.266 where there is no contribution from T1, Fig. 1C, D, blue) have the same features. The nitrogen features (Fig. 1C) From the above results the reaction of reduced NiR with excess NO gives T1 reduced T2 Cu 2+ -NO 2 − . However, the reaction of the reduced enzyme with limited NO has an EPR signal ( Fig. 2A, green), characteristic of a Cu + NO • species. 6 This species lacks d-d bands in the MCD spectrum (Fig. S3), further confirming that the Cu is reduced. Therefore, reduced NiR forms a Cu + NO • species at low concentrations of NO.The reaction of reduced NiR with excess NO involves two one-electron oxidati...