Figure 1: Overlay of FeSOD (orange ribbon) and MnSOD (magenta ribbon).
Figure 2: model for different Ems of Fe-substituted MnSOD (Fe(Mn)SOD) and Mn-substituted FeSOD (Mn(Fe)SOD.
Figure 3: Overlay of the active sites of FeSOD (white Cs), Fe(Mn)SOD (grey Cs), Q69H-FeSOD (green Cs) and Q69E-FeSOD (yellow Cs).
Figure 4: CD melting curves showing the increased stability of Q146E Mn-free MnSOD WT (Q146E-apoMn) compared to WT Mn-free MnSOD (WT-apoMn), which exceeds the stability of Mn-replete WT MnSOD (WT-holoMn).
Redox Tuning by ~ 1 V and the Metal Ion Requirement for Activity.
The Fe-SOD and Mn-SOD of E. coli have been manipulated to produce variants with reduction midpoint potentials ranging over 0.9 V (21 kCal/mol or 89 kJ/mol), without disruption of the active site [53,59]. Thus this system serves as a molecular laboratory for modifying a non-heme-Fe site to perform desired chemistry . Optical, EPR and NMR spectroscopy are used with Xray crystallography to elucidate the structures of these enzymes. Titrations of substrate analog binding, pH equilibria and redox equilibria provide quantitative measures of different fundamental elements of activity [16,42,52,53,57].
Thus we demonstrated that proteins can exploit control over the locations and energies of protons and hydrogen bonds to tune the Eos of proton-coupled redox reactions, over many 100s of mV [57,59].
Enzymes are catalysts extraordinaire. They can increase reaction rates by up to 21 orders of magnitude. They do this under mild conditions: room temperature and standard pressure, and they operate in a benign solvent: water. The most demanding reactions in biochemistry are mediated by redox-active enzymes: reactions such as conversion of N2 to NH3, and oxidation of CH4 to CH3OH. Nitrogenase breaks the triple bond of N2, the second strongest bond known, and it does so at room temperature in living cells. Thus, bound metal ions bring to life diverse and demanding chemistry that would not have been possible based on amino acids alone. The broad reactivity of metal ions however poses a challenge to the proteins than bind metal ions. The proteins have the task of both activating the metal for a particular reaction, yet preventing it from engaging in any other.
The Fe-specific and Mn-specific Superoxide Dismutases.
Superoxide dismutases (SODs) catalyze disproportionation of two molecules of superoxide producing one each of O2 and H2O2 via a reaction that involves alternation of the active site metal ion between the 3+ and the 2+ states . Thus they help prevent oxidative stress. SOD's mechanism involves control over activated oxygen and resolves two radical substrates. This mechanism of alternating oxidation and reduction requires that the enzyme reduction midpoint potential (Eo) be between those of the two half reactions and for optimal activity it should be intermediate [49,74].
The Fe-specific and Mn-specific Superoxide Dismutases.
Proteins specify the reactivity of their bound metal ions in part by determining their reduction potentials (the Eo: the energy associated with transfer of one electron). E. coli makes two different SODs, an Fe-specific SOD (FeSOD) and a Mn-specific SOD (MnSOD). These share 40% sequence identy and homologous overal structures. The active sites differ only very subtly, yet the Mn-SOD active site depresses the Eo by some 300 mV more than does the FeSOD site, regardless of whether Fe or Mn is bound [36,59]. Because the intrinsic Eo of Mn3+/2+ is several hundred mV higher than that of Fe3+/2+, the stronger redox depression of the Mn-specific protein brings the Eo of bound Mn close to that of Fe bound in the less depressing Fe-specific protein. Thus a requirement that the protein's redox tuning complement the metal ion's inate Eo suffices to explain the inactivity of Fe-substituted MnSOD and Mn-substituted FeSOD.
Origin of the 300 mV greater redox depression of the MnSOD protein.
The active sites of FeSOD and MnSOD are essentially superimposable. However we can understand their very different redox tuning in terms of the energy associated with proton transfer that is coupled to electron transfer (Mn3+OH- + e- +H+ ->Mn2+H2O). Thus, the MnSOD protein appears to suppress proton acquisition by the molecule of coordinated OH- that becomes H 2O as Mn3+ is reduced to Mn2+ (see second figure, at left) .
Redox tuning via coordinated solvent molecules or other exogenous ligands is a potentially widespread mechanism and offers very large flexibility for modification of the catalytic activity of existing enzymes, for new purposes.
Retuning the Eo of FeSOD by reversing the polarity of a hydrogen bond.
We proposed that a conserved active site Gln tunes the Eo of bound Fe by modulating the proton affinity of the coordinated solvent molecule, via the strength of H-bond donation from Gln to coordinated solvent. To test this proposal,we mutated the Gln to His and Glu, to provide a residue that could either donate or accept an H-bond (His), or a group that would be an obligate H-bond acceptor (Glu). As predicted, the Q69H mutant had a higher Eo than the WT and the Q69E mutant's Eo was higher still. Via a combination of tritrations, spectroscopy and X-ray crystallography, we showed that the 600 mV increase in Eo observed in the Q69E mutant can be explained by changes in the energy associated with redox-coupled proton transfer. Nonetheless, this large change in the reactivity of the Fe was achieved without disruption of the active site structure. Thus, we have not only engineered a significant change in reactivity but we have done so separately from substrate specificity.
Overall, including the Fe-substituted MnSOD, we have produced SOD variants with Eos ranging over 900 mV, in a conserved structural context .
The analogous Gln of MnSOD destabilizes the apoprotein structure by 35 oC compared to the Q146E mutant.
MnSODs contain a Gln analogous to the residue whose mutation changes FeSOD's Eo by 600 mV. It also has large effects on the stabilities of the apo- and metalo-protein complex. Mutation of Gln 146 to Glu increases the stability of the aproprotein by 35 oC compared to WT, but destabilizes the holoprotein . Thus the conserved Gln146 is proposed to strongly destabilize apoprotein as part of a mechanism that favours metal ion binding and prevents accumulation of non-metallated (inactive) SOD protein. A 35 oC increase in stability upon mutation of a single residue is a very dramatic result, and demonstrates the potency of negative selection (in this case against apo-protein).
Back to Home.
Coming soon:Group Members