Redox properties of human manganese superoxide dismutase and active-site mutants

The redox potential of human manganese superoxide dismutase (MnSOD) has bee n difficult to determine because of the problem of finding suitable electro n 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 b y 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 t he redox properties of the site-specific mutants His30Asn (H30N) and Tyr34P he (Y34F) and compared them with the wild-type enzyme. Crystal structures h ave shown that each mutation interrupts a hydrogen bond network in the acti ve site, and each causes a 10-fold decrease in the maximal velocity of cata lysis of superoxide dismutation as compared with wild type. The present stu dy shows that H30N and Y34F human MnSOD have very little effect, within exp erimental 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 Hi s30 and Tyr34 is more in support of catalysis, probably proton transport, a nd not in the tuning of the redox potential.