EPR−ENDOR of the Cu(I)NO Complex of Nitrite Reductase

Abstract

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).

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).…”

Enzyme catalysis captured using multiple structures from one crystal at varying temperatures

“…Extensive theoretical work has previously examined the binding of NO to T2Cu in CuNiRs and overall has proposed that end-on binding should be energetically preferred over the side-on binding observed in the crystal structures. ENDOR/ EPR experimental work on Rhodobacter sphaeroides CuNiR also suggested an end-on mode in solution (Usov et al, 2006). Our data thus suggest either that CuNiR in room-temperature solution and CuNiR in the crystalline state produce different product geometries, which we consider to be unlikely, or that the theoretical studies have not yet fully represented the binding geometry in vivo.…”

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

“…S2 in Supplementary Material), a fact that indicates that the features observed in the EPR signal cannot be attributed to interaction between the S = 1/2 radical species and the nuclear spin of the N atom of nitrite. The formation of copper(I)-nitrosyl adducts has been reported in a few copper complexes [45,46] and in the Nirs from A. faecalis S6 [41], R. sphaeroides [42], and A. cycloclastes [47]. The crystal structure of A. faecalis S6 Nir obtained by soaking ascorbate-reduced single crystals with NO-saturated buffer and that of as-isolated A. cycloclastes Nir with an endogenous NO ligand showed an unexpected side-on coordination of NO to Cu(I), in contrast to the end-on configuration adopted by NO in copper inorganic complexes [46].…”

“…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.…”