Replacement of the axial copper ligand methionine with lysine in amicyanin converts it to a zinc-binding protein that no longer binds copper

Abstract

The mutation of the axial ligand of the type I copper protein amicyanin from Met to Lys results in a protein that is spectroscopically invisible and redox inactive. M98K amicyanin acts as a competitive inhibitor in the reaction of native amicyanin with methylamine dehydrogenase indicating that the M98K mutation has not affected the affinity for its natural electron donor. The crystal structure of M98K amicyanin reveals that its overall structure is very similar to native amicyanin but that the type I binding site is occupied by zinc. Anomalous difference Fourier maps calculated using the data collected around the absorption edges of copper and zinc confirm the presence of Zn2+ at the type I site. The Lys98 NZ donates a hydrogen bond to a well-ordered water molecule at the type I site which enhances the ability of Lys98 to provide a ligand for Zn2+. Attempts to reconstitute M98K apoamicyanin with copper resulted in precipitation of the protein. The fact that the M98K mutation generated such a selective zinc-binding protein was surprising as ligation of zinc by Lys is rare and this ligand set is unique for zinc.

Zinc-mediated Allosteric Inhibition of Caspase-6

“…Although lysine can coordinate zinc, it has not been observed to coordinate other metals. For example, in the type I copper-binding protein amicyanin, the mutation of one of the copper ligands, methionine, to lysine converted the enzyme from copper binding to selectively zinc binding, demonstrating the specificity of lysine as a zinc ligand (62). Thus, the specificity of caspase-6 for inhibition by zinc may be the result of the rare ligand, Lys-36, participating in the coordination sphere.…”

A Conserved Acidic Residue in Phenylalanine Hydroxylase Contributes to Cofactor Affinity and Catalysis

“…Both the 3.0 and 1.95 Å crystal structures of ubiquitin (PDB entry 3H1U) 61 and lysine 63linked diubiquitin (PDB entry 2JF5) 62 reveal that one of the surface lysine residues of ubiquitin (Lys29) can coordinate cadmium (Figure 7 of the Supporting Information). Similar to the D139K mutant of cPAH described in the present study, artifactual lysine metal coordination has also been observed in other systems, such as by replacement of the coppercoordinating methionine in amicyanin with lysine, yielding an enzyme that binds zinc instead of copper (PDB entry 3RYM), 63 and by mutation of the axial heme iron methionine ligand in a bacterial cytochrome c 550 enzyme, leading to coordination of the amino group of lysine with the heme iron (PDB entry 2BH5). 64…”