Investigation of the Highly Active Manganese Superoxide Dismutase from Saccharomyces cerevisiae

Abstract

Manganese superoxide dismutase (MnSOD) from different species differs in its efficiency in removing high concentrations of superoxide (O₂⁻), due to different levels of product inhibition. Human MnSOD exhibits a substantially higher level of product inhibition than the MnSODs from bacteria. In order to investigate the mechanism of product inhibition and whether it is a feature common to eukaryotic MnSODs, we purified MnSOD from Saccharomyces cerevisiae (ScMnSOD). It was a tetramer with 0.6 equivalents of Mn per monomer. The catalytic activity of ScMnSOD was investigated by pulse radiolysis and compared with human and two bacterial (Escherichia coli and Deinococcus radiodurans) MnSODs. To our surprise, ScMnSOD most efficiently facilitates removal of high concentrations of O₂⁻ among these MnSODs. The gating value k₂/k₃ that characterizes the level of product inhibition scales as ScMnSOD > D. radiodurans MnSOD > E. coli MnSOD > human MnSOD. While most MnSODs rest as the oxidized form, ScMnSOD was isolated in the Mn²⁺ oxidation state as revealed by its optical and electron paramagnetic resonance spectra. This finding poses the possibility of elucidating the origin of product inhibition by comparing human MnSOD with ScMnSOD

Manganese superoxide dismutase (MnSOD) enzymes catalyze superoxide (O₂⁻) disproportionation by a mechanism that is more complex than those of the other SODs. In particular the reduction of superoxide can proceed via one of two pathways. One pathway dominates when the O₂⁻ concentration is low relative to the enzyme concentration (reaction 2) and the other pathway dominates when the ratio [O₂⁻]:[MnSOD] is high (reactions 3 and 4). The paradoxical finding is that MnSOD is a less effective SOD catalyst when O₂⁻ levels are elevated.¹

(1)

(2)

(3)

(4)

The depressed catalytic activity at high O₂⁻ concentrations is known to be due to the formation of a product-inhibited Mn³⁺-peroxo adduct resulting from the inner sphere oxidation of Mn²⁺SOD by O₂⁻ (reaction 3). This pathway is considerably slower than the outer sphere oxidation and protonation pathway described in reaction 2. The relative levels of product inhibition are described kinetically by the value of k₂/k₃ for different MnSODs (Table I). The contribution from this slower pathway is particularly pronounced in human MnSOD.¹ Thus the O₂⁻ removal and H₂O₂ production rates are dependent on the relative levels of MnSOD and O₂⁻, and the degree of product inhibition of the specific MnSOD present.

Table I.

Rate constants for the different MnSODs

Organism	k1 (nMs−1)	k2 (nMs−1)	k3 (nMs−1)	k4 (s−1)	k2/k3
Human8b	1.4	0.6	0.5	130	1.6
E. coli8c	1.1	0.9	0.17	60	5.3
D. radiodurans8a	1.2	1.1	0.07	30	16
S. cerevisiae (this work)	1.1 – 1.5	0.8	0.04 – 0.05	90 –140	16 – 20

Superoxide concentrations are known to be variable in cells; for example, it has recently been shown that transient O₂⁻ bursts, termed “superoxide flashes”, are formed in human mitochondria,² creating the possibility of even greater variability in H₂O₂ formation rates. However, slower product-inhibited pathway in human MnSOD would allow for more constant H₂O₂ formation even when O₂⁻ concentrations vary.

Low levels of H₂O₂ play an important role in signaling in mammalian cells, regulating numerous processes including rates of cell growth and division.³ It has been proposed that the slower pathway for human MnSOD appeared in response to an evolutionary pressure to control more tightly intracellular H₂O₂ levels,¹ to reduce H₂O₂ mediated oxidative damage and to optimize its signaling function.⁴ The k₂/k₃ values determined for human and bacterial MnSODs (Table I) are consistent with this hypothesis. However, structural studies of different MnSODs, both wild type and mutant, have yet to reveal why k₂/k₃ differs so dramatically for this enzyme.¹

The budding yeast Saccharomyces cerevisiae is widely used as a single-cell model for higher eukaryotic organisms because it is remarkably similar to mammalian cells. S. cerevisiae also appears to be less sensitive to H₂O₂ than human cells, and the only currently known H₂O₂ sensing proteins in S. cerevisiae (YAP1p and Skn7p) are involved in regulating oxidative stress protection; a more general signaling role has yet to be found.^3,5 Both human MnSOD and S. cerevisiae MnSOD (ScMnSOD) are tetramers⁶ and localized to the mitochondrial matrix,⁷ while most bacterial MnSODs are dimers. Moreover, human MnSOD shares greater sequence similarity with ScMnSOD than with Escherichia coli or Deinococcus radiodurans MnSODs (62.2%, 52.9%, and 54.5% respectively).⁸ Published reports of the activity of ScMnSOD, do not include a determination of the degree of product inhibition.^6,9 We therefore turned our attention to characterizing the catalytic mechanism of ScMnSOD with the expectation that the high contribution from the product-inhibited pathway would prove to be a property common to eukaryotic MnSODs. Surprisingly, we found instead that ScMnSOD is even less product-inhibited than the bacterial MnSODs characterized to date, surpassing even the high activity of MnSOD from the radiation resistant bacterium D. radiodurans.^8a

The gene of ScMnSOD,⁹ which includes the mitochondrial targeting sequence, was inserted into the plasmid YEp352. The enzyme was overexpressed in S. cerevisiae and purified using a protocol modified from that of Fridovich et al. (Supporting Information).⁶ The protein was isolated as a tetramer with the leader sequence removed and contained 0.6 equivalents of Mn per monomer, as per size exclusion chromatography, mass spectrometry, and ICP-MS, respectively.

Although the fitting of the kinetic data reported here matches the experimental decay of pulsed O₂⁻, out of the four rate constants, only k₂ has been measured directly by pulse radiolysis. k₂ was measured by oxidizing the resting enzyme with substoichiometric amounts of O₂⁻ and following the appearance of Mn³⁺SOD, which has a characteristic absorption band near 480 nm (Supporting Information). Unlike other MnSODs, ScMnSOD was isolated in the reduced state, precluding direct measurement of k₁. k₁, k₃ and k₄ were determined by fitting the observed rate of O₂⁻ (ε₂₆₀=2000 M⁻¹cm⁻¹) loss at multiple enzyme concentrations (1–10 µM) and initial O₂⁻ concentrations (2–48 µM) using PRWIN, conditions that, while not physiological, are necessary to study kinetics by pulse radiolysis.^1,8 The rate constants are compared to those known for other MnSODs in Table I.

The ability of ScMnSOD¹⁰ to catalyze the dismutation of O₂⁻ was found to exceed that of the other characterized MnSODs. At low concentrations of O₂⁻ ([O₂⁻] ≈ [MnSOD]), the observed rates of disappearance are similar for all the four enzymes (Fig. 1A). However at high concentrations ([O₂⁻] >> [MnSOD]), the differences in activities are pronounced (Fig. 1B). At the highest concentrations of O₂⁻ employed in pulse radiolysis, the order of the activities of the enzymes is S. cerevisiae > D. radiodurans > E. coli > human, which is identical to the ordering of k₂/k₃, indicating that the deciding factor is the tendency to form the product-inhibited state.

Figure 1.

The decay of 2.3-µM (A) and 48-µM (B) O₂⁻ concentrations with different types of MnSOD. ScMnSOD pulse radiolysis data are shown in black, while the lines are from Kintecus¹⁰ computer modeling using rate constants in Table I: MnSOD from D. radiodurans (purple); E. coli (green); and human (blue); all at 1 µM Mn.

Most known MnSODs rest in the Mn³⁺ oxidation state,¹¹ but our ScMnSOD was consistently isolated as predominantly reduced Mn²⁺SOD; the electronic absorption spectrum of as-isolated ScMnSOD lacked the visible absorption band with a maximum near 480 nm. It was reported previously that the optical absorption spectrum of ScMn³⁺SOD was unusual relative to those of other MnSODs,⁹ but the spectrum of ScMn³⁺SOD that we obtained by reaction of as-isolated ScMnSOD with O₂⁻ using pulse radiolysis (Fig. 2A, dots) was very similar to those of other Mn³⁺SODs, with an extinction coefficient of ~800 M⁻¹cm⁻¹. The small measured absorbance at 480 nm (Fig. 2A, line) corresponds to greater than 90% of the as-isolated enzyme being in the Mn²⁺ state.

Figure 2.

ScMnSOD was isolated in the Mn²⁺ oxidation state. A) Electronic absorption spectra of as-isolated ScMnSOD (line) and Mn³⁺SOD obtained after reaction with O₂⁻ in pulse radiolysis (dots). B) EPR spectrum of as-isolated ScMnSOD. Instrumental parameters: temperature, 4.7 K; microwave frequency, 9.685 GHz; microwave power, 0.2 mW; modulation amplitude, 0.8 mT.

The EPR spectrum of our as-isolated ScMnSOD also indicates that the enzyme is reduced, since the perpendicular-mode EPR spectrum is similar to those of other Mn²⁺SODs,¹² with the usual six-line hyperfine splitting from the ⁵⁵Mn nucleus (I = 5/2) seen at g_eff = 6.0 (Fig 2B). We looked for evidence of the integer spin Mn³⁺ (S = 2) by parallel-mode EPR, but our spectrum lacked the sextet hyperfine pattern typically displayed by Mn³⁺SOD (Supporting Information).¹³

The only other MnSOD enzymes that have been isolated in the Mn²⁺ oxidation state are mutant MnSODs, most notably the Gln143 mutants of the human enzyme.¹⁴ However, the factors that determine the resting oxidation state are unknown.

Increased levels of MnSOD activity have been shown to slow down tumor growth in cultured human cells and in animal studies¹⁵, and it has been proposed that this effect is related to cellular H₂O₂ levels. For that reason and to improve our understanding of the basis of the observed product-inhibition, human MnSOD has been repeatedly mutated in attempts to make its activity resemble that of the bacterial proteins, but with limited success.^1,8c As described above, human MnSOD already shares greater sequence similarity with ScMnSOD than with the bacterial enzymes, and the two eukaryotic proteins are tetramers while the bacterial ones are dimers. Also, ScMnSOD resembles the bacterial ones in that k₃ is small, but is similar to the human enzyme in that k₄ is large. Thus, to improve our understanding of what causes the unusual kinetic properties of human MnSOD, it may be more productive to compare/contrast it with ScMnSOD than with the bacterial MnSODs. Investigation of the slight structural differences between the enzymes may provide a key to understanding the chemical mechanism of product inhibition. We will also continue to study the evolutionary significance of product inhibition by studying MnSOD from other organisms.