The Side-On Copper(I) Nitrosyl Geometry in Copper Nitrite Reductase Is Due to Steric Interactions with Isoleucine-257

Abstract

Density functional theory calculations were used to investigate the binding mode of copper(I) nitrosyl (Cu(I)-NO) in copper nitrite reductase (CuNIR). The end-on Cu(I)-NO geometry (2) was found to be the global energy minimum, while the side-on binding mode (1) corresponds to a local minimum. Isoleucine-257 severely interacts sterically with the Cu(I)-NO unit when bound end-on but not in the side-on case. In addition, the side-on geometry is also stabilized by a hydrogen bond between aspartic acid-98 and NO, estimated to be approximately 3 kcal/mol. The steric constraint of the CuNIR active site is mainly responsible for the observed side-on coordination of NO in the CuNIR crystal structure. We speculate that a small conformational change of the active site that slightly changes the position of isoleucine-257 would allow NO to bind end-on. This explains the observed end-on binding of NO to copper(I) when CuNIR is in solution.

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

“…Third, cryogenic manipulations for reducing radiation damages in SRX have also been focused as a factor that changes the population of amino acid residues (33, 34) and enzyme-substrate complexes (35). Crystallographic (36), computational (37), and spectroscopic (38-40) studies actually show that binding modes of NO 2 − and NO in CuNiR crystal structures can differ from those in physiological environments.…”

“…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).…”

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

“…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]. Distinct DFT studies showed that interactions with amino acid side chains are responsible for causing the unusual side-on orientation in Nirs [46,[48][49][50][51]. Copper-nitrosyl adducts present S = 1/2 ground state associated with nearly axial EPR signals (g iso~2 ) and with fast relaxing behavior [46].…”

Copper Active Sites in Biology

“…Recent calculations by Merkle and Lehnert which include the second sphere residues Asp98 and Ile289 show that the side-on bound geometry is a local minimum while the end-on geometry is the global minimum, with a small barrier of 1.0 kcal/mol required for conversion from the side-on to the end-on structure. 1267 Interactions that contribute to stabilizing the side-on coordination geometry include hydrogen bonding from Asp98, which is 1–2 kcal/mol stronger in the side-on case, and steric constraints from Ile289, which restricts the geometry of the end-on NO complex. Thus, the side-on coordination of the CuNiR NO complex observed in crystals may represent a metastable form that relaxes to end-on coordination in solution when the positions of the sidechains of Ile289 and Asp98 are more flexible.…”