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. 2006 Oct 15;41(8):1338-50.
doi: 10.1016/j.freeradbiomed.2006.07.015. Epub 2006 Jul 21.

A new paradigm: manganese superoxide dismutase influences the production of H2O2 in cells and thereby their biological state

Garry R Buettner  1 , Chin F Ng, Min Wang, V G J Rodgers, Freya Q Schafer
Affiliations

A new paradigm: manganese superoxide dismutase influences the production of H2O2 in cells and thereby their biological state

Garry R Buettner et al. Free Radic Biol Med. .

Abstract

The principal source of hydrogen peroxide in mitochondria is thought to be from the dismutation of superoxide via the enzyme manganese superoxide dismutase (MnSOD). However, the nature of the effect of SOD on the cellular production of H(2)O(2) is not widely appreciated. The current paradigm is that the presence of SOD results in a lower level of H(2)O(2) because it would prevent the non-enzymatic reactions of superoxide that form H(2)O(2). The goal of this work was to: a) demonstrate that SOD can increase the flux of H(2)O(2), and b) use kinetic modelling to determine what kinetic and thermodynamic conditions result in SOD increasing the flux of H(2)O(2). We examined two biological sources of superoxide production (xanthine oxidase and coenzyme Q semiquinone, CoQ(*-) that have different thermodynamic and kinetic properties. We found that SOD could change the rate of formation of H(2)O(2) in cases where equilibrium-specific reactions form superoxide with an equilibrium constant (K) less than 1. An example is the formation of superoxide in the electron transport chain (ETC) of the mitochondria by the reaction of ubisemiquinone radical with dioxygen. We measured the rate of release of H(2)O(2) into culture medium from cells with differing levels of MnSOD. We found that the higher the level of SOD, the greater the rate of accumulation of H(2)O(2). Results with kinetic modelling were consistent with this observation; the steady-state level of H(2)O(2) increases if K<1, for example CoQ(*-)+O(2)-->CoQ+O(2)(*-). However, when K>1, e.g. xanthine oxidase forming O(2)(*-), SOD does not affect the steady state-level of H(2)O(2). Thus, the current paradigm that SOD will lower the flux of H(2)O(2) does not hold for the ETC. These observations indicate that MnSOD contributes to the flux of H(2)O(2) in cells and thereby is involved in establishing the cellular redox environment and thus the biological state of the cell.

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Figures

Figure 1
Figure 1. The flow of electrons through xanthine oxidase
Xanthine makes a complex with XO at the molybdenum site (ka = 6 × 106 M−1 s−1, 37°C [116]); then xanthine is oxidized to urate with reduction of the molybdenum (kb = 15 s−1). This is followed by transfer of electrons to the Fe/S centers (kc = 8.5 × 103 s−1 [117]); then to FAD (kd = 2 × 102 s−1 [117]), and finally production of O2•− from FADH• (k1a = 7 × 104 M−1 s−1). The thermodynamics for the formation of O2•− from the FADH• indicate an equilibrium constant of ≈104. Thus the back reaction would have a rate constant of ≈7 M−1 s−1 [30].
Figure 2
Figure 2. CoQ•− of the ETC can make O2•−, a source of H2O2
CoQ•− can be formed during electron/proton transfer during the protonmotive Q-cycle, from comproportionation of CoQ and CoQH2, and also by other one-electron oxidation-reduction processes. K1b is the equilibrium constant highlighted in the model. (kc and kd are the rate constants for comproportionation and disproportionation, respectively).
Figure 3
Figure 3. The rate of accumulation of H2O2 in the media increases with the cellular MnSOD activity
(♦) The H2O2 released from MCF-7 cells and several clones overexpressing MnSOD (1.1-, 3-, 6-, and 19-fold) in exponential growth was determined at several time points over a period of two hours. The concentrations of H2O2 in the medium were measured using Amplex red. The ordinate represents the change in the [H2O2] in the media versus time compared to wildtype MCF-7 cells (1 on the left ordinate scale represents 2.8 zmol cell−1 s−1). Results are from three independent experiments ± standard deviation. ( ) The mitochondrial steady-state level of H2O2 (nM) from the kinetic model achieved with different levels of MnSOD (0.7–10 μM) as presented in Figure 4D.
Figure 4
Figure 4. The steady-state concentration of H2O2 in cells can be a function of the level of MnSOD
The kinetic model clearly demonstrates that when K < 1 for Rxn 1 the level of SOD will determine the flux of H2O2 as well as its steady-state level. (A) K= 1000; (B) K= 10; (C) K= 0.1; (D) K= 0.001. The different values for the equilibrium constant were achieved by varying the rate constant for the reverse reaction of Rxn 1, keeping the value of the forward rate constant at 8 × 103 M−1 s−1. In the kinetic model, the capacity to remove H2O2 was kept constant.
Figure 4
Figure 4. The steady-state concentration of H2O2 in cells can be a function of the level of MnSOD
The kinetic model clearly demonstrates that when K < 1 for Rxn 1 the level of SOD will determine the flux of H2O2 as well as its steady-state level. (A) K= 1000; (B) K= 10; (C) K= 0.1; (D) K= 0.001. The different values for the equilibrium constant were achieved by varying the rate constant for the reverse reaction of Rxn 1, keeping the value of the forward rate constant at 8 × 103 M−1 s−1. In the kinetic model, the capacity to remove H2O2 was kept constant.
Figure 4
Figure 4. The steady-state concentration of H2O2 in cells can be a function of the level of MnSOD
The kinetic model clearly demonstrates that when K < 1 for Rxn 1 the level of SOD will determine the flux of H2O2 as well as its steady-state level. (A) K= 1000; (B) K= 10; (C) K= 0.1; (D) K= 0.001. The different values for the equilibrium constant were achieved by varying the rate constant for the reverse reaction of Rxn 1, keeping the value of the forward rate constant at 8 × 103 M−1 s−1. In the kinetic model, the capacity to remove H2O2 was kept constant.
Figure 4
Figure 4. The steady-state concentration of H2O2 in cells can be a function of the level of MnSOD
The kinetic model clearly demonstrates that when K < 1 for Rxn 1 the level of SOD will determine the flux of H2O2 as well as its steady-state level. (A) K= 1000; (B) K= 10; (C) K= 0.1; (D) K= 0.001. The different values for the equilibrium constant were achieved by varying the rate constant for the reverse reaction of Rxn 1, keeping the value of the forward rate constant at 8 × 103 M−1 s−1. In the kinetic model, the capacity to remove H2O2 was kept constant.
Figure 5
Figure 5. The ability of SOD to modulate the flux of H2O2 and [H2O2]ss depends on the availability of a sufficient pool of electrons
The ETC has a large pool of electrons. The vast majority flow through complex IV to O2 forming H2O at the end of the ETC; a very small fraction form O2•−. The CoQ-system has appropriate thermodynamics and access to a large pool of electrons so that MnSOD can modulate the flux of O2•− and subsequently H2O2. XO has unfavorable thermodynamics, which do not allow SOD to modulate the flux of O2•−; in addition there is a very limited pool of electrons to draw from. Although CoQ•−is thought to be the principal source for formation of superoxide in mitochondria, other sites may also be sources of O2•−.
Figure 6
Figure 6. MnSOD serves as a rheostat for both superoxide and hydrogen peroxide signalling
By controlling [O2•−]ss MnSOD influences certain reactive Fe-centers in enzymes and proteins that are sensitive to oxidation/reduction by O2•−. By changing the flux of H2O2, MnSOD will also modulate signalling processes that go through the thiol-system. Thus, MnSOD serves as a rheostat for both one-electron and two-electron signalling pathways. These signalling processes set the redox environment of cells, which in turn establishes the biological state of cells and tissues.
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