Mutation of tyrosine 34 to phenylalanine eliminates the active site pK of reduced iron-containing superoxide dismutase.

We have compared the magnetic resonance properties and pH dependence of wild-type and mutant Fe-containing superoxide dismutase (Fe-SOD) in which the conserved active site tyrosine (Tyr 34) is replaced by phenylalanine. The EPR spectrum of the oxidized state and the NMR spectrum of the paramagnetically shifted resonances of the reduced state indicate that in both states the active site is relatively unperturbed by the mutation. Similarly, the mutant Fe-SOD retains approximately 41% of wild-type catalytic activity on a per Fe basis. However, replacement of Tyr 34 by Phe abolishes both NMR spectroscopic signatures of the active site pK of 8.5 of (reduced) Fe2+-SOD. Neither accessibility to base-catalyzed exchange nor the chemical shifts of active site residues are affected by pH in the range of 6.5-10.5 in Y34F Fe2+-SOD. Thus, the active site pK of 8.5 of Fe2+-SOD most likely corresponds to deprotonation of Tyr 34. The widespread chemical shift changes associated with the pK could reflect Tyr 34's participation in the active site hydrogen bonding network and the network's propagation of the effects of deprotonating Tyr 34 to the Fe2+. Deprotonation of Tyr 34 can also explain the dramatic decrease in active site accessibility to base-catalyzed exchange as the result of electrostatic repulsion between the exchange catalyst OH- and the (Tyr 34)- ion formed at high pH. Similar electrostatic repulsion between (Tyr 34)- and the substrate O2.- is also consistent with the observed increase in KM above pH 9.