STRUCTURAL STUDIES OF THE BLUE COPPER PROTEINS

HELEN RUTH ENGESETH, Purdue University

Abstract

Model studies using Ni(II), Co(II), and Zn(II) as metal centers have been prepared from the Schiff's base adducts of o-mercaptobenzaldehyde and two amines, 2,5-diamino-2,5-dimethylhexane and t-butylamine. The resulting complexes present N(,2)S(,2) donor sets and steric constraints which favor pseudotetrahedral coordination geometries. With the guide of these systems, the near infrared transitions of the Ni(II) derivative of azurin have been located, confirming a nonplanar geometry occurs in the protein. The ('113)Cd NMR spectra of spinach plastocyanin, stellacyanin, Alcaligenes faecalis and Pseudomonas aeruginosa azurins have been measured. The stellacyanin and azurins give resonances very nearly the same, indicating a similar coordination environment. Results indicate a change in coordination number may occur in plastocyanin relative to the others. The relaxation mechanism is dominated by chemical shift anisotropy. The resonance of the Pseudomonas aeruginosa azurin displays a pH dependence that can be assigned to the small change in the protein conformation that occurs with the protonation of His-35. Differential scanning calorimetry has been used to study the thermally induced denaturation of the metal-free and various metal-containing derivatives of Pseudomonas aeruginosa azurin, stellacyanin and spinach plastocyanin. The azurin and stellacyanin derivatives show remarkable similarities in their calorimetric behavior and differ significantly from that observed in the corresponding plastocyanin derivatives. The transitions observed for the azurins and stellacyanins deviate markedly from a two-state model. Metal ion binding increases the stability of all the proteins (relative to the apoprotein), though the increase is much more dramatic in the azurin and stellacyanin systems. The apparent (DELTA)G (relative to the denatured form) has been calculated at 298 K and from these, approximate binding constants for the metals studied have been calculated. Determination of the entropic contribution to the relative free energy of the metalloproteins indicate that metal ion association in many cases results in structures of high internal order relative to the apoproteins.

Degree

Ph.D.

Subject Area

Chemistry

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