KINETIC-STUDIES OF SUPEROXIDE DISMUTASES - PROPERTIES OF THE MANGANESE-CONTAINING PROTEIN FROM THERMUS-THERMOPHILUS

The catalysis of superoxide dismutation (2O2- + 2H+ --> H2O2 + O2) by manganese superoxide dismutase (MnSOD) from Thermus Thermophilus was examined by stopped-flow spectrophotometry. As found earlier by McAdam et al. [McAdam, M.E.; Fox, R.A.; Lavelle, F.; Fielden, E.M. Biochem J. 1977, 165, 81-87], decay curves of O2- in the presence of MnSOD from Bacillus Stearothermophilus are characterized by three distinct phases: rapid disappearance of O2- (the ''burst'' phase), a period of approximately zero-order disappearance of O2- (the ''steady-state'' phase), and a very rapid depletion of O2- toward the end of the reaction. The enzyme from T. Thermophilus shows a similar kinetic pattern, and our data provide a chemical explanation for this behavior: The molar consumption of O2- in the burst phase is ([O2-]B/[Mn]T) approximately 80. The magnitude of the burst is decreased approximately 2.5-fold in D2O, whereas the zero-order phase is the same in both solvent. This indicates that proton transfer is probably the rate-limiting step when the enzyme is saturated with O2- and that the reaction by which inactive enzyme returns to active enzyme is not limited by proton transfer. At low temperatures (2-6-degrees-C) in D2O, the overall reaction was sufficiently slow to allow observation of spectral changes associated with the metal chromophore during the steady state, and we were able to obtain an absorption spectrum of the enzyme during this period. This was assigned to the inactive form of the enzyme and is characterized by a band near 650 nm (epsilon approximately 230 [Mn]-1 cm-1) and a band near 410 nm (epsilon approximately 700 [Mn]-1 cm-1). We speculate that inactivation of the enzyme occurs by oxidative addition of O2- to Mn(II), within a Michaelis complex, forming a cyclic peroxo complex of Mn(III) with the reverse of this reaction yielding active enzyme.