Novel Biological Copper Proteins through Anion Addition to the Mutant Met121Gly of Pseudomonas aeruginosa Azurin

Abstract

Azide, cyanide, and thiocyanate are the anions that, on addition to a variant produced by site‐directed mutagenesis, Met121Gly, of Pseudomonas aeruginosa azurin, produce unprecedented copper proteins with markedly different spectroscopic properties. This ability to generate ligand‐accessible metal centers in coordinatively saturated metalloproteins by directed mutagenesis might possibly bring the synthesis of metalloproteins that can be used as ion sensors a step nearer.

Engineering a bifunctional copper site in the cupredoxin fold by loop-directed mutagenesis

“… 58 The variants described herein are able to bind either imidazole or azide, with higher binding affinities than those reported for azurin loop mutants. 63 , 66 These results suggest that the bifunctional nature of T1 sites can be further expanded.…”

The Mutation Met121→His Creates a Type‐1.5 Copper Site in Alcaligenes denitrificans Azurin

“…In recent studies Romero et al, 1993 ;Kroes et al, 1995;Vidacovic and Germanas, 1995) of a number of Met121 mutants of azurin, the majority of the Cu sites could be classified as type-I sites. However, Cu sites that resulted from the replacement of the axial methionine by Lys, Glu or His were found to have spectroscopic properties between those of type-1 and type-2 sites Pascher et al, 1993;Kroes et al, 1995).…”

A Rearranging Ligand Enables Allosteric Control of Catalytic Activity in Copper-containing Nitrite Reductase

“…Earlier this approach was used to investigate the type-1 copper site in azurin. [12][13][14][15][16][17][18][19] By using the enzyme nitrite reductase, it is possible to monitor the electron transfer function, which is connected with the catalytic activity. For NiR, we earlier found 20 that when this approach was applied to the C-terminal histidine ligand, catalytic activity was lost because the midpoint potential of the type-1 site was altered too much, also in the presence of external ligands.…”

“…This question was not addressed in earlier reports. 18,29,30 We replaced the original type-1 methionine ligand in NiR by a glycine (M150G) to create space for an external ligand such as an imidazole or an alcohol. As controls for these ligands we prepared the M150H and M150T variants.…”