The dynamic properties of the M121H azurin metal site as studied by NMR of the paramagnetic Cu(II) and Co(II) metalloderivatives.
The M121H azurin mutant in solution presents various species in equilibrium that can be detected and studied by 1H NMR of the Cu(II) and Co(II) paramagnetic metalloderivatives. In both cases up to three species are observed in slow exchange, the proportions of which are different for the two metalloderivatives. Above pH 5 the major species displays a tetrahedral coordination in which the His121 can be observed as a coordinated residue. Its metal site corresponds to a new type of site that is defined as a type 1.5 site. The second and third species resemble the wild type (type 1) azurin and, above pH 4.5, they are present only at a low concentration. At low pH a protonation process increases the proportion of both type 1 species at the expense of the type 1.5 species. This process, characterized by a pKa = 4.3, is assigned to the protonation of His121. At high pH the NMR spectrum of the Co(II)-M121H azurin experiences an additional transition, which is not observed in the case of the Cu(II) protein. The dynamic properties of the M121H metal site appear to be related to changes in the coordination geometry and the strength of the axial interaction between the Ndelta1 (His121) and the metal.
Salgado
J
J
Leiden Institute of Chemistry, University of Leiden, P.O. Box 9502, 2300 RA Leiden, The Netherlands.
Kroes
S J
SJ
Berg
A
A
Moratal
J M
JM
Canters
G W
GW
D001400:Azurin / Q000737:chemistry* / Q000378:metabolism; D003035:Cobalt / Q000378:metabolism*; D003300:Copper / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D013056:Spectrophotometry, Ultraviolet; D013696:Temperature
D001400:Azurin; D003035:Cobalt; D003300:Copper