The pH dependence of intramolecular electron transfer rates in sulfite oxidase at high and low anion concentrations

The individual rate constants for intramolecular electron transfer (IET) be tween the (MoFeII)-Fe-VI and (MoFeIII)-Fe-V forms of chicken liver sulfite oxidase (SO) have been determined at a variety of pH values, and at high an d low anion concentrations. Large anions such as EDTA do not inhibit IET as dramatically as do small anions such as SO42- and Cl-, which suggests that specific anion binding at the sterically constrained Mo active site is nec essary for IET inhibition to occur.IET may require that SO adopt a conforma tion in which the Mo and Fe centers are held in close proximity by electros tatic interactions between the predominantly positively charged Mo active s ite, and the negatively charged heme edge. Thus, small anions which can fit into the Mo active site will weaken this electrostatic attraction and disf avor IET. The rate constant for IET from Fe-II to Mo-VI decreases with incr easing pH, both in the presence and absence of 50 mM SO42-. However, the ra te constant for the reverse process exhibits no significant pH dependence i n the absence of SO42-, and increases with pH in the presence of 50 mM SO42 -. This behavior is consistent with a mechanism in which IET from Mo-V to F e-III is coupled to proton transfer from Mo-V-OH to OH-, and the reverse IE T process is coupled to proton transfer from H2O to Mo-VI=O. At high concen trations of small anions, direct access of H2O or OH- to the Mo-OH will be blocked, which provides a second possible mechanism for inhibition of IET b y such anions. Inhibition by anions is not strictly competitive, however, a nd Tyr322 may play an important intermediary role in transferring the proto n when an anion blocks direct access of H2O or OH- to the Mo-OH. Competing H-bonding interactions of the Mo-OH moiety with Tyr322 and with the anion o ccupying the active site may also be responsible for the well-known equilib rium between two EPR-distinct forms of SO that is observed for the two-elec tron reduced enzyme.