Redox properties of human manganese superoxide dismutase and active-site mutants.
The redox potential of human manganese superoxide dismutase (MnSOD) has been difficult to determine because of the problem of finding suitable electron mediators. We have found that ferricyanide and pentacyanoaminoferrate can be used as electron mediators, although equilibration is very slow with a half-time near 6 h. Values of the midpoint potential were determined both by allowing enzyme and mediators to equilibrate up to 38 h and by reductive titration adding dithionite to enzyme and mediator. An overall value of the midpoint potential was found to be 393 +/- 29 mV. To elucidate the role of His30 and Tyr34 in the active site of human MnSOD, we have also measured the redox properties of the site-specific mutants His30Asn (H30N) and Tyr34Phe (Y34F) and compared them with the wild-type enzyme. Crystal structures have shown that each mutation interrupts a hydrogen bond network in the active site, and each causes a 10-fold decrease in the maximal velocity of catalysis of superoxide dismutation as compared with wild type. The present study shows that H30N and Y34F human MnSOD have very little effect, within experimental uncertainty, on the redox potential of the active-site metal. The redox potentials determined electrochemically were 365 +/- 28 mV for H30N and 435 +/- 30 mV for Y34F MnSOD. These results suggest that the role of His30 and Tyr34 is more in support of catalysis, probably proton transport, and not in the tuning of the redox potential.
Lévêque
V J
VJ
Department of Pharmacology, and Neuroscience, University of Florida, Gainesville, Florida 32610-0267, USA.
Vance
C K
CK
Nick
H S
HS
Silverman
D N
DN
D001665:Binding Sites; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins; D013482:Superoxide Dismutase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017186:Titrimetry
D011994:Recombinant Proteins; D013482:Superoxide Dismutase