Mutation of Tyrosine 34 to Phenylalanine Eliminates the Active Site pK of Reduced Iron-Containing Superoxide Dismutase

Abstract

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.

Anion Binding Properties of Reduced and Oxidized Iron-Containing Superoxide Dismutase Reveal No Requirement for Tyrosine 34

“…The pK near 9 affecting the oxidized state of WTFe 3+ SOD reflects coordination of OH -to Fe 3+ , but the pK of 8.5 affecting the reduced state reflects ionization of Tyr34 (21,25). Indeed, we find that the oxidized state pK persists in Y34F-Fe 3+ SOD, but the reduced state pK is absent (21). Although the overall V max of WT-FeSOD does not change, the K m increases at high pH (6,22).…”

Characterization of the Product-inhibited Complex in Catalysis by Human Manganese Superoxide Dismutase

“…The crystal structure of Y34F Mn-SOD shows that its activesite structure is nearly exactly superimposable with that of the wild type except for the absence of the side-chain hydroxyl of residue 34 (8). The mutants Y34F Mn-SOD and Y34F Fe-SOD have been observed in a number of studies comparing their catalytic and spectral properties to the wild type (7)(8)(9)(10). The catalytic activity of these mutants is decreased as much as 10-fold.…”

Interrupting the Hydrogen Bond Network at the Active Site of Human Manganese Superoxide Dismutase

“…This protonation promotes the dissociation of product from the enzyme and perhaps decreases the extent of product inhibition. Recent studies using site-specific mutagenesis have clarified the role of Tyr 34 in the active site of MnSOD showing that replacement of this residue with Phe leaves a mutant with considerable catalytic activity (5)(6)(7)(8). Tyr 34 in human MnSOD has properties consistent with its participation in the proton transfer events reported by k cat ; the replacement of Tyr 34 with Phe in human MnSOD resulted in the decrease in k cat by an order of magnitude with no change in k cat /K m (8).…”

“…1. This network has been identified by crystallography (8,10) and by the changes in proton NMR chemical shifts for many active-site residues in reduced E. coli iron superoxide dismutase caused by the replacement of Tyr 34 with Phe (7). In this network the solvent ligand of manganese is hydrogen-bonded to the side chain of Gln 143 , which in turn forms a hydrogen bond with the phenolic hydroxyl of Tyr 34 .…”