The catalysis of superoxide dismutation ( 2 0 1 + 2H+ -H202 + 0,) 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 01 in the presence of MnSOD from Bacillus Stearothermophilus are characterized by three distinct phases: rapid disappearance of 02-(the "burst" phase), a period of approximately zero-order disappearance of 02-(the "steady-state" phase), and a very rapid depletion of 02-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 0; in the burst phase is ([O2-Ie/[MnlT) -80. The magnitude of the burst is decreased -2.5-fold in D20, whereas the zero-order phase is the same in both solvents. This indicates that proton transfer is probably the rate-limiting step when the enzyme is saturated with 0; and that the reaction by which inactive enzyme returns to active enzyme is not limited by proton transfer. At low temperatures (2-6 "C) in D20, 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 (c -230 [Mnl-' cm-I) and a band near 410 nm (c -700 [Mnl-' cm-I). We speculate that inactivation of the enzyme occurs by oxidative addition of 0; to Mn(II), within a Michaelis complex, forming a cyclic peroxo complex of Mn(II1) with the reverse of this reaction yielding active enzyme. k j -650 S-' Mn":02-+ Mn"':o22k-5 -10 s-' A reaction scheme composed of a cyclic redox process, as described previously for the FeSOD of Escherichia coli [Bull, C . ; Fee, J. A. J . Am. Chem. SOC. 1985, 107, 3295-33041, and the above reversible side reaction adequately account for the kinetic behavior of MnSODs.In previous communications we described the steady-state kinetic properties of the iron-containing superoxide dismutase from E. coli,' of Cu/Zn-containing superoxide dismutase (SOD35) from bovine tissues,* and of FeEDTA.3v35 In this paper we describe our studies with the manganese-containing protein from Thermus thermophilus.There are three types of superoxide dismutases as determined by the metal involved in the catalysis of reaction 1: Cu, Fe, and Mn. The general properties, distribution, and possible biological (1) function of these proteins have been discussed in a large number of review and discussion papers (cf. Ref 1 of ref 1 for reviews). The present work is concerned with manganese-containing superoxide dismutases, and the object of study is the protein from Thermus thermophilus. This protein is a tetramer of 21 kDa subunits, each of which binds, on the average, -0.6 Mn(II1) ions. Solutions of the protein have a reddish-pur...
