Structural reorganization of the copper binding site involving Thr15 of mavicyanin from Cucurbita pepo medullosa (Zucchini) upon reduction

Mavicyanin, a glycosylated protein isolated from Cucurbita pepo medullosa (zucchini), is a member of the phytocyanin subfamily containing one polypeptide chain of 109 amino residues and an unusual type-I Cu site in which the copper ligands are His(45), Cys(98), His(91), and Gln(96). The crystal structures of oxidized and reduced mavicyanin were determined at 1.6 and 1.9 angstrom resolution, respectively. Mavicyanin has a core structure of seven polypeptide beta-strands arranged as a beta-sandwich organized into two beta-sheets, and the structure considerably resembles that of stellacyanin from cucumber (CST) or cucumber basic protein (CBP). A flexible region was not observed on superimpositioning of the oxidized and reduced mavicyanin structures. However, the Cu-II-epsilon-O-Gln(96) bond length was extended by 0.47 angstrom, and the Thr(15) residue was rotated by 60.0 degrees and O-gamma 1-Thr(15) moved from a distance of 4.78 to 2.58 angstrom from the ligand Gln(96) forming a new hydrogen bond between O-gamma 1-Thr(15) and epsilon-O-Gln(96) upon reduction. The reorganization of copper coordination geometry of mavicyanin upon reduction arouses reduction potential decreased above pH 8 [Battistuzzi et al. (2001) J. Inorg. Biochem. 83, 223-227]. The rotation of Thr(15) and the hydrogen bonding with the ligand Gln(96) may constitute structural evidence of the decrease in the reduction potential at high pH.