Electronic Characterization of the Oxidized State of the Blue Copper Protein Rusticyanin by ¹H NMR:  Is the Axial Methionine the Dominant Influence for the High Redox Potential?

Abstract

The oxidized state of rusticyanin, the blue copper protein with the highest redox potential in its class, has been investigated through (1)H nuclear magnetic resonance applied to its cobalt(II) derivative. The assignment of the protons belonging to the coordinated residues has been performed. Many other amino acids situated in the vicinity of the metal ion, including six hydrophobic residues (isoleucine140 and five phenylalanines) have also been identified. The orientation of the main axes of the magnetic susceptibility tensor for the cobalt(II)-rusticyanin as well as its axial, Deltachi(ax), and rhombic, Deltachi(rh), magnetic susceptibility anisotropy components have been determined. A comparison of the present results with those previously obtained for cobalt(II)azurin [Donaire, A., Salgado, J., Moratal, J. M. (1998) Biochemistry 37, 8659-8673] allows us to provide further insights into the reasons for the high redox potential of this protein. According to our results, the interaction between the metal ion and the thioether Sdelta of the axial methionine is not as influential as the strong destabilizing effect that the hydrophobic residues close to the metal ion undergo in the oxidized state.

Metal-ligand interactions in perturbed blue copper sites: a paramagnetic 1H NMR study of Co(II)-pseudoazurin

“…The pseudocontact contribution to the chemical shifts was estimated by assuming the same axial and rhombic magnetic susceptibility tensor components determined for Co(II)-rusticyanin [37]. Initially, the tensor orientation was assumed to be similar to the one found for Co(II)-rusticyanin, but during calculations the x,y plane of the magnetic tensor was rotated in steps of 10°around the z-axis to explore the possible effects of different orientations.…”

“…Paramagnetic NMR investigations on cupredoxins include studies of the native Cu(II) species [19,20,21,22,23] and of their Co(II) and Ni(II) derivatives [15,24,25,26,27,28,29,30,31,32,33,34,35,36,37]. These studies have afforded evidence on the interplay between the strength of the metal-axial ligand interaction and the metal-Cys covalence [15,20,21].…”

“…Tetragonally perturbed blue copper centers (pseudoazurin, rusticyanin, plantacyanin and nitrite reductase) present a different scenario, since the axial Met ligand is generally closer to Cu(II) than in classic sites, but the main perturbation arises from a different orientation of the Cys and Met ligands with respect to the His residues [16,38]. The only reported NMR study on these perturbed centers corresponds to the Co(II) derivative of rusticyanin, where the orientation and components of the susceptibility magnetic tensor are available [37]. Additional studies on other blue copper proteins with tetragonally perturbed sites may be helpful to understand the influence of a tetragonally distorted geometry on the structural and electronic properties of the metal center.…”

Active Site Comparison of CoII Blue and Green Nitrite Reductases

“…This is confirmed [23,27] [e] From Thiobacillus ferrooxidans at pH 6.0 and 20 8C including the d pc and d c contributions to d obs . [29] [f] From spinach at 30 8C and pH 7.8. [31] [g] From Paracoccus versutus at 40 8C and pH 8.0 (the His96 N e2 H resonance was observed at 22 8C and pH 5.0).…”

“…[52] More sizable changes upon the addition of nitrite could be anticipated at even lower pH values, however the T1Cu I /T2Co II NiR derivatives were not stable under these conditions. [27] which has a classic T1 copper site, and Co II rusticyanin (RST), [29] which possesses a distorted center (see Table 3). [27] ).…”