Proton-coupled electron transfer in Fe-superoxide dismutase and Mn-superoxide dismutase

Fe-containing superoxide dismutase (FeSOD) and MnSOD are widely assumed to employ the same catalytic mechanism. However this has not been completely tested. In 1985, Bull and Fee showed that FeSOD took up a proton upon reduction [J. Am. Chem. Soc. 107 (1985) 3295]. We now demonstrate that MnSOD incorporates the same crucial coupling between electron transfer and proton transfer. The redox-coupled H⁺ acceptor has been presumed to be the coordinated solvent molecule, in both FeSOD and MnSOD, however this is very difficult to test experimentally. We have now examined the most plausible alternative: that Tyr34 accepts a proton upon SOD reduction. We report specific incorporation of ¹³C in the C^ζ positions of Tyr residues, assignment of the C^ζ signal of Tyr34 in each of oxidized FeSOD and MnSOD, and direct NMR observations showing that in both cases, Tyr34 is in the neutral protonated state. Thus Tyr34 cannot accept a proton upon SOD reduction, and coordinated solvent is concluded to be the redox-coupled H⁺ acceptor instead, in both FeSOD and MnSOD. We have also confirmed by direct ¹³C observation that the pK of 8.5 of reduced FeSOD corresponds to deprotonation of Tyr34. This work thus provides experimental proof of important commonalities between the detailed mechanisms of FeSOD and MnSOD.