Resolution of the Spectroscopy versus Crystallography Issue for NO Intermediates of Nitrite Reductase from Rhodobacter sphaeroides

Abstract

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...

Redox-coupled proton transfer mechanism in nitrite reductase revealed by femtosecond crystallography

“…A conundrum remained: Which Cu-NO species is produced in the enzymatic cycle (VI)? Whereas side-on NO is stabilized in crystal structures (22,23,25), spectroscopic and computational studies indicate that end-on NO is a physiological intermediate (37)(38)(39)(40). Although visualization of an end-on NO species with short lifetime has been difficult, time-resolved SFX may enable it (60,61).…”

Demonstration of Proton-coupled Electron Transfer in the Copper-containing Nitrite Reductases

“…xylosoxidans (NCIMB 11015, AxNiR) was overexpressed in Escherichia coli BL21(DE3) cells, and the purification was similar to the method described previously (42,43) (see supplemental material for detailed description). AxNiR concentrations were determined using an extinction coefficient of 4 Very recently spectroscopic and computational methods have shown that ET from the T1 to the T2Cu sites in the green copper enzyme RsNiR (Rhodobacter sphaeroides) requires the initial proton transfer from the Asp residue in the active site pocket to nitrite thereby triggering the reductive cleavage of the substrate (40,41 ⑀ 595 ϭ 6.3 mM Ϫ1 cm Ϫ1 (9,18). The AxNiR enzyme lacking the copper atom in the T2Cu center (T2DNiR) was expressed and purified in the same way as AxNiR except that the dialysis steps into CuSO 4 were omitted (27,42,44).…”

“…The relevance of these active site residues, however, as well as the timing of the two protonation steps is still a matter of debate (35,40,41). 4 There are no experimental studies that have been aimed at directly examining the kinetic coupling of PT and ET steps in AxNiR.…”

Overexpression, purification, and biochemical and spectroscopic characterization of copper-containing nitrite reductase from Sinorhizobium meliloti 2011. Study of the interaction of the catalytic copper center with nitrite and NO

“…This interpretation was based on EPR simulations assuming typical g-and A-values for a T2 site plus hyperfine coupling to four N-nuclei at g ⊥ , three from the coordinating histidines and one from the NO, and on X-ray data which showed a side-on NO coordinated to Cu [41]. In contrast, other authors, on the basis of EPR, ENDOR (Electron-Nuclear Double Resonance), and DFT (Density Functional Theory) calculations in R. sphaeroides NiR [42,43], concluded that the species generated in solution of ascorbate-reduced Nir with excess NO corresponds to a Cu II -NO 2 − species.…”