Some Notes Added in Proof

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

“…Thus, mutation of Tyr34 increases the binding site's capacity, enabling it to bind a triatomic substrate analogue, not just the monatomic F -that binds to WT. The K d is at least 40 times smaller than that of WT (>100 mM), indicating that Tyr34 imposes a similar net steric cost on N 3 -binding in both oxidation states. Thus, the reduced state is similar to the oxidized state in that mutation of Tyr34 to Phe weakens binding of small substrates (loss of an H-bond in the absence of steric penalty) but strengthens binding of larger substrates (subject to strong steric penalty).…”

“…On the basis of optically monitored titrations and eqs 4 and 5, we find that a single N 3 -inhibits Y34F-FeSOD with a 20-fold lower K I than its K I for WTFeSOD (28), and 20-fold tighter N 3 -binding for Y34F-Fesubstituted-MnSOD than WT-Fe-substituted-MnSOD (9). Tighter binding to Y34F-Fe 3+ SOD of the larger-thansubstrate analogue N 3 -is likely due to relief of steric repulsion between Tyr34's OH and N 3 -.…”

“…In the cases of the other titrations, where the weak binding approximation holds, the simplification [L] ) L T could be made. Thus eq 6 was used for N 3 -binding to WT-Fe 3+ SOD and eq 7 was used to describe F -binding to two sites after correcting the absorbance data for dilution.…”

“…The active site 1 H NMR spectrum of (oxidized) Fe 3+ SOD is much less resolved than that of (reduced) Fe 2+ SOD, due to the slower electronic spin relaxation of Fe 3+ , which causes faster proton relaxation (3,36). These paramagnetically shifted resonances' line widths are dominated by the paramagnetic contribution to the transverse proton relaxation rate (1/T 2 ) p , which in Fe 3+ SOD is expected to be proportional to the electron spin relaxation time.…”

“…Since high pH supresses F -binding, the data were fit to the following competitive binding model: mutually exclusive deprotonation of an ionizable group and binding of F -(Scheme 1). 3 Data were fit with eqs 8a-c, in which the observed chemical shift (δ obs ) represents a population-weighted average (Table 1) and the pH was raised by adding KOH. The active site 1 H NMR spectrum has been shown to be the same at pH 6 and 7 (24), so the differences between the top and middle spectra represent the effects of F -.…”