The mutation Met121-->His creates a type-1.5 copper site in Alcaligenes denitrificans azurin.
The Cu ligand Met121 in azurin of Alcaligenes denitrificans was mutated to His. The spectroscopic and mechanistic properties of [M121H]azurin appear to be pH dependent with a pKa of 3.8 due to the ionization of His121. The [M121H]azurin mutant exhibits two major distinct metal-site-coordination geometries which coexist in solution according to pH-dependent equilibrium. Both species have been spectroscopically characterized by ultraviolet-visible, EPR and resonance Raman spectroscopies. At neutral pH, His121 is deprotonated and acts as the fourth ligand of the Cu; the spectroscopic characteristics of the Cu site at this pH are halfway between those of a type-1 and a type-2 Cu site, and the site is referred to as a type-1.5 or intermediate Cu site. The spectral data are compatible with a tetrahedral geometry of this site. At low pH, the spectroscopic data indicate that [M121H]azurin has a trigonal type-1 rhombic Cu site.
Kroes
S J
SJ
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
Hoitink
C W
CW
Andrew
C R
CR
Ai
J
J
Sanders-Loehr
J
J
Messerschmidt
A
A
Hagen
W R
WR
Canters
G W
GW
D000421:Alcaligenes / Q000737:chemistry*; D001400:Azurin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry / Q000378:metabolism*; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D013057:Spectrum Analysis; D013059:Spectrum Analysis, Raman
D008024:Ligands; D001400:Azurin; D006639:Histidine; D003300:Copper; D008715:Methionine