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?

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, Delta chi (ax), and rhombic, Delta chi (rh), magnetic susceptibility anisotropy components have been determined. A comparison of the present results with those previously obtained for cobalt(Il)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 S delta 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.