An investigation of Cu2Zn2 superoxide dismutase and its Ile-137 mutant at high pH.
The activity profile of the Cu2Zn2HSOD Ile-137 mutant has a pKa of 9.6, i.e. one unit lower than the wild type (WT). This property has allowed us to investigate the inactive high pH form of the enzyme before denaturation occurs. The electronic and EPR spectra do not change with the above pKa. The 1H NMR spectrum of the Cu2Co2-analog reveals slight decreases in the hyperfine shifts of the protons of His-48 at high pH, which are consistent with a water molecule becoming closer to the copper ion, as detected through water 1H T-1(1) NMR measurements. The affinity of azide at high pH is lower than at low pH, though still sizeable. The WT follows the same pattern up to pH congruent to pKa. It appears that the drop in activity is not related to any major change involving the metal coordination sphere, but is related to changes in the electrostatic potential due to the deprotonation process.
Banci
L
L
Department of Chemistry, University of Florence, Italy.
Bertini
I
I
Turano
P
P
D001386:Azides / Q000737:chemistry; D002942:Circular Dichroism; D003035:Cobalt / Q000737:chemistry; D003300:Copper / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007532:Isoleucine / Q000737:chemistry*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D013053:Spectrophotometry; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014867:Water / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D001386:Azides; D007532:Isoleucine; D014867:Water; D003035:Cobalt; D006639:Histidine; D003300:Copper; D013482:Superoxide Dismutase; D015032:Zinc