A new type 2 copper cysteinate azurin. Involvement of an engineered exposed cysteine in copper binding through internal rearrangement.
The double mutant H117G/N42C azurin exhibits tetragonal type 2 copper site characteristics with Cys(42) as one of the copper ligands as concluded from spectroscopic evidence (UV-visible, EPR, and resonance Raman). Analysis of the kinetics of copper uptake by the apoprotein by means of stopped flow spectroscopy suggests that the solvent-exposed Cys(42) assists in binding the metal ion and carrying it over to the active site where it becomes coordinated by, among others, a second cysteine, Cys(112). A structure is proposed in which the loop from residue 36 to 47 has rearranged to form a tetragonal type 2 copper site with Cys(42) as one of the ligands. The process of copper uptake as observed for the double mutant may be relevant for a better understanding of the way copper chaperones accept and transfer metal ions in the living cell.
van Amsterdam
Irene M C
IM
Leiden Institute of Chemistry, Gorlaeus Laboratories, University of Leiden, Einsteinweg 55, P. O. Box 9502, The Netherlands.
Ubbink
Marcellus
M
van den Bosch
Marieke
M
Rotsaert
Frederik
F
Sanders-Loehr
Joann
J
Canters
Gerard W
GW
D001400:Azurin / Q000737:chemistry*; D001665:Binding Sites; D003300:Copper / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry*; D004574:Electromagnetic Fields; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman; D013997:Time Factors; D014466:Ultraviolet Rays
D008024:Ligands; D001400:Azurin; D003300:Copper; D003545:Cysteine