pH dependences are different between the FeSODs and the

During normal catalytic turnover, the active site must take up two protons and thus the activity of the Fe/MnSOD family is dependent on pH at high pH ranges. At low pH ranges, the active site can readily take up a proton upon reduction (Bull & Fee, 1985). At high pH levels, the activity of both FeSODs and MnSODs is decreased relative to the physiological pH ranges. It was shown that the overall Km of Ec-FeSOD was

independent of pH below pH 8.0 but above this pH range Km increases in a pH-

dependent manner (Bull & Fee, 1985). Above pH 10 for every pH unit increase there is a tenfold increase in Km. Similar results were seen in Ec-MnSOD (Miller et al., 2003).

active site. There is a number of pH-dependent events associated with protonation/deprotonation and association/disassociation of hydroxide ions at the active site, which may differ between MnSODs and FeSODs, as summarised by Maliekal and co-workers (Maliekal et al., 2002). There are differences in pH dependence of spectroscopic properties between the FeSODs and MnSODs, the most notable difference being a difference of one pH unit for the mid-range pK of Mn2+SOD compared to Fe2+SOD. This may merely be a consequence of tuning of redox potentials for the FeSODs and MnSODs.

The location of the proton taken up at the active site upon reduction in Fe3+SODs (Bull & Fee, 1985) and Mn3+SODs (Miller et al., 2003) is unknown. However, it is unlikely to be the solvent-access funnel tyrosine, as shown by NMR experiments (Miller et al., 2003). It is widely believed that the fifth ligand, the coordinated solvent molecule that is ascribed as an hydroxide in both FeSODs and MnSODs, is one of the proton acceptors. There is a marked difference between Fe3+SODs and Mn3+SODs when it comes to pH dependence (Maliekal et al., 2002). In Ec-Fe3+SOD the coordination number changes from five to six when moving from pH 7 to pH 10 as shown by X-ray absorption spectroscopy (Tierney et al., 1995). There is a widely observed pK for Ec-Fe3+SOD at 8.5 (Bull & Fee, 1985, Tierney et al., 1995, Miller et al., 2003), which is interpreted as an hydroxide ion coordinating to the active-site metal ion as the sixth ligand rather than deprotonation of a residue in the solvent-access funnel (Miller et al., 2003); refer to Figure 2-6. However, once reduced, Ec-Fe2+SOD undergoes a pH-dependent transition with a similar mid-point pK of 8.4, which is believed to be the deprotonation of the conserved tyrosine.

Figure 2-6. Redox- and pH-dependent changes in active-site structure of FeSOD.

Figure 2-6. At physiological pH range, the oxidised form of the metallo-enzyme has a penta-coordinate Fe3+ cofactor, which binds to an hydroxide ion. The hydroxide ion is stabilised by interactions with two hydrogen-bonding partners, the metal-ligating aspartate and a nearby glutamine. Upon reduction of the active- site metal ion, a proton is also taken up converting the coordinated hydroxide to a water molecule. Both the oxidised and reduced forms undergo pH-linked transitions with pK of ~8.5. In the oxidised form this is associated with hydroxide entering the active site and in the reduced form this is associated with the de- protonation of the conserved tyrosine. At high pH, active-site metal-ion reduction is associated with the uptake of a proton and the loss of a water molecule. This mechanism is based on various pK determinations from several sources (Bull & Fee, 1985, Tierney et al., 1995, Sorkin et al., 1997, Sorkin & Miller, 1997).

H2O H+ e- _{+ H}+ e- _{+ H}+ pK₌

8.4

8.5

There is also marked pH dependence in MnSODs with the pK of oxidised Mn3+SOD being 8.5 (Miller et al., 2003, Maliekal et al., 2002, Whittaker & Whittaker, 1997a). There are currently two hypotheses concerning the nature of this pH-dependent event. Some believe that this corresponds to the deprotonation of the solvent-access funnel tyrosine in MnSODs (Jackson et al., 2002, Guan et al., 1998, Maliekal et al., 2002); refer to Figure 2-7. The alternate, but slightly older, hypothesis is that this pK is due to an hydroxide ion associating with the active-site Mn (Whittaker & Whittaker, 1997a, Borgstahl et al., 2000), similar to FeSODs. The higher pK of 10.3 shows pH dependence in Mn2+SOD (Maliekal et al., 2002, Whittaker & Whittaker, 1997a), where HFEPR shows a transition (Tabares et al., 2006), indicating that the pH-dependent transitions are altered, but not eliminated, by changes in redox state, unlike the FeSODs where the pK are comparable for both oxidised and reduced states.

Both of these hypotheses are based on Ec-MnSOD and rely on interpretation of spectroscopic and structural data. Tyrosine deprotonation is mainly supported by 13C NMR of labelled tyrosines (Miller et al., 2003, Maliekal et al., 2002). The most compelling evidence for hydroxide association is the existence at low temperature of a hexa-coordinate structure for Ec-MnSOD (Borgstahl et al., 2000).

Complementary to this is research done on the pH dependence of the binding of a sixth ligand to the Fe form of the cambialistic SOD from Propionibacterium shermanii (Meier et al., 1998). The structure has been solved at four different pH including some above and below the pH where coordination changes from five to six (Schmidt, 1999). The occupancy of the sixth ligand, a hydroxide, increases as pH increases and is mimicked by the binding of the small-molecule inhibitor fluoride, which binds with partial occupancy in neutral pH ranges.

Figure 2-7. Temperature-, redox- and pH-dependent changes in active-site structure of MnSOD. H+ HXO e- _{+ H}+ pK₌

9.6

10.5

Figure 2-7. At low physiological pH ranges, the oxidised form of MnSOD contains a penta-coordinated Mn3+ ion that has a coordinated hydroxide that is stabilised by hydrogen bonds to the metal ligand aspartate and to a glutamine. At these pH ranges, the nearby tyrosine is protonated. In the oxidised form of the metallo-enzyme, the deprotonation of the tyrosine occurs with a pK of 9.6. Upon reduction, a proton is taken up by the coordinated hydroxide converting it to a water molecule. In the reduced form of MnSOD, the deprotonation of the conserved tyrosine occurs with a pK of 10.5. Under cryogenic conditions there is a transition to a hexa-coordinate state where HXO (a water or hydroxide ion),

binds to the Mn3+.