Reactions of H(2)O(2) with superoxide dismutase were studied by e.p.r. (electron paramagnetic resonance) spectroscopy and other methods. In agreement with earlier work, the Cu(2+) of the enzyme is reduced by H(2)O(2), although the reaction does not go to completion and its kinetics are not simple. With dilute enzyme the time for half-reduction with 9mm-H(2)O(2) is about 150ms. It is suggested that the reaction is a one-electron reduction, involving liberation of O(2) (-). On somewhat more prolonged exposure to H(2)O(2), the enzyme is inactivated. For enzyme in dilute solution and over a limited range of H(2)O(2) concentrations, inactivation is first-order with respect to enzyme and reagent, with k=3.1m(-1).s(-1) at 20-25 degrees C. Inactivation is accompanied by marked changes in the e.p.r. and visible spectra and appears to be associated with destruction of one histidine residue per subunit. It is suggested that this histidine is close to the metal in the native enzyme and essential for its enzymic activity.

1. Detailed studies on the mechanism of the enzymic reaction of bovine superoxide dismutase were carried out by using pulse radiolysis and electron paramagnetic resonance (e.p.r.). 2. The second-order rate constant for reaction between superoxide dismutase and the superoxide ion was redetermined as (2.37+/-0.18)x10(9)m(-1).s(-1) at 25 degrees C. This reaction governs the turnover, and any first-order steps must have rate constants higher than about 10(6)s(-1). Turnover has a low activation energy and is slowed substantially when the viscosity is increased with glycerol, confirming that the reaction rate is near the limit for diffusion control. In water a reversible conformation change to a less active form appears to take place above about 40 degrees C. 3. Pre-steady-state rates of reduction and reoxidation of copper in the enzyme are consistent with these processes being rate-limiting in enzyme turnover. 4. Examination, with the help of computer simulation, of the e.p.r. spectra at 9 and 35GHz of native superoxide dismutase indicated that, apart from 10-20% of impurities, only one species of Cu(2+) is distinguishable. Further, the specific activity of our enzyme preparations, measured by pulse radiolysis, is at least as high as that obtained by other workers. 5. Nevertheless, measurement of the proportion of copper present as Cu(2+) (determined both optically and by e.p.r. spectroscopy) in the steady states approached from both the oxidized and the reduced forms of the enzyme, indicates (after allowing for the impurities) that only half of the copper atoms participate in turnover. E.p.r. spectroscopy provided no evidence for differences between functioning and non-functioning Cu(2+) atoms. 6. It is suggested that the results may be best interpreted in terms of an allosteric type of mechanism, with two initially indistinguishable copper atoms in the enzyme. Reaction of one of these with a superoxide ion then renders the other, at least transiently, unreactive.

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Superoxide radical ions (O2-) produced by the radiolytic reduction of oxygenated formate solutions and by the xanthine oxidase-catalysed oxidation of xanthine were shown to oxidize the haem groups in oxyhaemoglobin and reduce those in methaemoglobin as in reactions (1) and (2): (see articles) Reaction (1) is suppressed by reaction (8) when [O2-]exceeds 10 muM, but consumes all the O2- generated in oxyhaemoglobin solutions when [oxyhaemoglobin] greater than 160 muM and [O2-]less than 1 nM at pH 7. The yield of reaction (2) is also maximal in methaemoglobin solutions under similar conditions, but less than one haem group is reduced per O2- radical. From studies of (a) the yield of reactions (1) and (2) at variable [haemoglobin] and rates of production of O2-, (b) their suppression by superoxide dismutase, and (c) equilibria observed with mixtures of oxyhaemoglobin and methaemoglobin, it is shown that k1/k2=0.7 +/- 0.2 and k1 = (4 +/- 1) X 10(3) M-1-S-1 At pH7, and k1 and k2 decrease with increasing pH. Concentrations and rate constants are expressed in terms of haem-group concentrations. Concentrations of superoxide dismutase observed in normal erythrocytes are sufficient to suppress reactions (1) and (2), and hence prevent the formation of excessive methaemoglobin.

The mechanism of the enzymic reaction of an iron-containing superoxide dismutase purified from the marine bacterium Photobacterium leiognathi was studied by using pulse radiolysis. Measurements of activity were done with two different preparations of enzyme containing either 1.6 or 1.15g-atom of iron/mol. In both cases, identical values of the second-order rate constant for reaction between superoxide dismutase and the superoxide ion in the pH range 6.2-9.0 (k = 5.5 x 108M-' s-at pH 8.0) were found. As with the bovine erythrocuprein, there was no evidence for substrate saturation. The effects of reducing agents (H202, sodium ascorbate or CO2-radicals) on the visible and the electron-paramagnetic-resonance spectra of the superoxide dismutase containing 1.6g-atom of ferric iron/mol indicate that this enzyme contains two different types of iron. Turnover experiments demonstrate that only that fraction of the ferric iron that is reduced by H202 is involved in the catalysis, being alternately oxidized and reduced by O2-; both the oxidation and the reduction steps have a rate constant equal to that measured under turnover conditions. These results are interpreted by assuming that the superoxide dismutase isolated from the organism contains 1 g-atom of catalytic iron/mol and a variable amount of non-catalytic iron. This interpretation is discussed in relation to the stoicheiometry reported for iron-containing superoxide dismutases prepared from several other organisms.

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In today's world we appear to place a premium on happiness. Happiness is often portrayed, directly or indirectly, as one of the key aims of education. To suggest that education is concerned with promoting unhappiness or even despair would, in many contexts, seem outlandish. This paper challenges these widely held views. Focusing on the work of the great Russian writer, Fyodor Dostoevsky, I argue that despair, the origins of which lie in our reflective consciousness, is a defining feature of human life. Education, I suggest, should not be seen as a flight from despair but as a process of deepening our understanding of suffering and its potentially pivotal role in our humanisation. In developing these ideas, I draw on Kierkegaard's The Sickness Unto Death and Unamuno's The Tragic Sense of Life in Men and Nations, among other sources.

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