FIgure 1: cartoon of the active site of MnSOD.

Figure 2: model for different E_ms 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).

Anne-Frances Miller

Director, University of Kentucky NMR spectroscopy facility

Associate Professor of Chemistry, and of Biochemistry.Download CV

Redox tuning, intermediates in O₂ activation and redesign of an active site for desired activity.

Redox-Active Metalloenzymes. Enzymes are catalysts extraordinaire. They can increase reaction rates by up to 19 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 tripple bond of N3, 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. 

Explaining the metal-ion specificity of activity of FeSOD and MnSOD from E. coli. Proteins specify the reactivity of their bound metal ions in part by determining their reduction potentials (the Em: 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 reduction midpoint potential (Em) by some 300 mV more than does the FeSOD site, regardless of whether Fe or Mn is bound (**). This suffices to explain the inactivity of Fe-substituted MnSOD and Mn-substituted FeSOD.

We can explain the 300 mV greater redox depression of the MnSOD protein in terms of the energy associated with proton transfer that is coupled to electron transfer (M³⁺•OH^- + e^- +H⁺ ->M²⁺•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 exogenous ligands is a potentially widespread mechanism and offers very large flexibility for modification of the catalytic activity of existing enzymes, for new purposes.  Click here for references.  

Retuning the reduction midpoint potential of FeSOD by reversing the polarity of a hydrogen bond. We proposed that a conserved active site Gln tunes the E_m 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, or a group that would be an obligate H-bond acceptor. As predicted, the Q69H mutant had a higher E_m than the WT and the Q69E mutant's E_m was higher still. Via a combination of tritrations, spectroscopy and X-ray crystallography, we find that the 600 mV increase in E_m 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 desired chainge in reactivity but we have done so separately from substrate specificity. Overall, including the Fe-substituted MnSOD, we have produced SOD variants with E_ms ranging over 900 mV, in a conserved structural context. Click here for references.

Trapping Fe-bound activated O₂ species. FeSOD's active site beaers strong resemblance to the non-heme Fe active sites of a large group of enzymes involved in waste detoxification, antibiotic biosynthesis, hormone metabolism and protein maturation reactions. While the reactions and substrates are very diverse, the common theme is activation of O₂ , whose triplet ground state presents a kinetic barrier to otherwise extremely thermodynamically favorable reaction. We are using our mis-tuned FeSOD active sites to allow binding and trapping of O₂ , its activated superoxide form, or the product peroxide. Thus we can learn about the electronics and structures of these crucial intermediates in the reactions of so many enzymes. Click here to learn more.