Paramagnetic 1H NMR spectrum of nickel(II) pseudoazurin: investigation of the active site structure and the acid and alkaline transitions.
The paramagnetic (1)H NMR spectrum of Ni(II) pseudoazurin [(PA)Ni(II)] possesses a number of resonances exhibiting sizable Fermi-contact shifts. These have been assigned to protons associated with the four ligating amino acids, His40, Cys78, His81, and Met86. The shifts experienced by the C(gamma)H protons of the axial Met86 ligand are unprecedented compared to other Ni(II)- and Co(II)-substituted cupredoxins (the C(gamma)(1)H signal is found at 432.5 ppm at 25 degrees C). The large shift of protons of the axial Met86 ligand highlights a strong Ni(II)-S(Met) interaction in (PA)Ni(II). The paramagnetic (1)H NMR spectrum of (PA)Ni(II) is altered by decreasing and increasing the pH value from 8.0. At acidic pH a number of the hyperfine-shifted resonances undergo limited changes in their chemical shift values. This effect is assigned to the surface His6 residue whose protonation results in a structural modification of the active site. Increasing the pH value from 8.0 has a more significant effect on the paramagnetic (1)H NMR spectrum of (PA)Ni(II), and the alkaline transition can now be assigned to two surface lysine residues close to the active site of the protein. The effect of altering pH on the (1)H NMR spectrum of Ni(II) pseudoazurin is smaller than that previously observed in the Cu(II) protein indicating more limited structural rearrangements at the non-native metal site.
Dennison
Christopher
C
Department of Chemistry, University of Newcastle upon Tyne, UK. christopher.dennison@ncl.ac.uk
Sato
Katsuko
K