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.
Leiden Institute of Chemistry, Gorlaeus Laboratories, University of Leiden, Einsteinweg 55, P. O. Box 9502, The Netherlands.
Irene M C
IM
van Amsterdam
Marcellus
M
Ubbink
Marieke
M
van den Bosch
Frederik
F
Rotsaert
Joann
J
Sanders-Loehr
Gerard W
GW
Canters
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