RACK-INDUCED METAL-BINDING VS. FLEXIBILITY - MET121HIS AZURIN CRYSTAL-STRUCTURES AT DIFFERENT PH

The rack-induced bonding mechanism of metals to proteins is a useful c oncept for explaining the generation of metal sites in electron transf er proteins, such as the blue copper proteins, that are designed for r apid electron transfer, The trigonal pyramidal structure imposed by th e protein with three strong equatorial ligands (one Cys and two His) p rovides a favorable geometry for both cuprous and cupric oxidation sta tes, However, the crystal structures of the Met121His mutant of azurin from Alcaligenes denitrificans at pH 6.5 (1.89- and 1.91-Angstrom res olutions) and pH 3.5 (2.45-Angstrom resolution) show that the preforme d metal binding cavity in the protein is more flexible than expected, At high pH (6.5), the Cu site retains the same three equatorial ligand s as in the wild-type azurin and adds His121 as a fourth strong ligand , creating a tetrahedral copper site geometry with a green color refer red to as 1.5 type, In the low pH (3.5) structure, the protonation of His121 causes a conformational change in residues 117-123, moving His1 21 away from the copper, The empty coordination site is occupied by an oxygen atom of a nitrate molecule of the buffer solution, This axial ligand is coordinated less strongly, generating a distorted tetrahedra l copper geometry with a blue color and spectroscopic properties of a type-1 site, These crystal structures demonstrate that blue copper pro teins are flexible enough to permit a range of movement of the Cu atom along the axial direction of the trigonal pyramid.