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

Abstract

The individual rate constants for intramolecular electron transfer (IET) between the Mo(VI)Fe(II) and Mo(V)Fe(III) forms of chicken liver sulfite oxidase (SO) have been determined at a variety of pH values, and at high and low anion concentrations. Large anions such as EDTA do not inhibit IET as dramatically as do small anions such as SO4(2-) and Cl-, which suggests that specific anion binding at the sterically constrained Mo active site is necessary for IET inhibition to occur.IET may require that SO adopt a conformation in which the Mo and Fe centers are held in close proximity by electrostatic interactions between the predominantly positively charged Mo active site, and the negatively charged heme edge. Thus, small anions which can fit into the Mo active site will weaken this electrostatic attraction and disfavor IET. The rate constant for IET from Fe(II) to Mo(VI) decreases with increasing pH, both in the presence and absence of 50 mM SO4(2-) . However, the rate constant for the reverse process exhibits no significant pH dependence in the absence of SO4(2-), and increases with pH in the presence of 50 mM S04(2-). This behavior is consistent with a mechanism in which IET from Mo(V) to Fe(III) is coupled to proton transfer from Mo(V)-OH to OH-, and the reverse IET process is coupled to proton transfer from H2O to Mo(VI) = O. At high concentrations 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 by such anions. Inhibition by anions is not strictly competitive, however, and Tyr322 may play an important intermediary role in transferring the proton 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 occupying the active site may also be responsible for the well-known equilibrium between two EPR-distinct forms of SO that is observed for the two-electron reduced enzyme.

Viscosity Effects on Eukaryotic Nitrate Reductase Activity

“…NADH:ferricyanide reductase activity of CbR showed a similar result, leading to the conclusion that no large conformational change occurred when the flavin of the enzyme was reduced and transferred electrons directly to an acceptor in solution. Neither of these catalytic reactions involves IET, and the absence of a viscosity effect is consistent with the concept established in studies of the impact of viscosity on IET in SOX (13,14). NaR, similar to SOX, utilizes IET in catalysis of the NAD(P)H:NaR reaction and partial reactions such as Cyt c reduction.…”

“…Furthermore, it is clear from the impact of solution viscosity on ferricyanide reductase activity that IET is the rate-limiting process in this very rapid catalytic activity of NaR. These results are similar to those found for other redox enzymes with IET from or to a Cyt b domain, such as flavocytochrome b 2 and SOX (13,14,31). Thus, the movement of Cyt b domain relative to CbR is gating the electron and proton transfer processes in MoR and holo-NaR (Scheme 2).…”

“…Recent studies of mammalian SOX kinetics showed that the IET rate was impacted by solution viscosity (13,14), confirming the hypothesis that the Cyt b domain was undergoing a large conformational change during catalysis relative to the Mo-MPT-containing sulfite-oxidizing module (9). NaR has a similar structure to SOX in that the Cyt b domain is tethered to the Mo-MPT-containing nitrate-reducing module via a flexible "hinge" sequence.…”

“…1 and Table I), the interpretation of the results is clearly more difficult than for enzymes with a single flexible hinge region such as mammalian SOX and yeast flavocytochrome b 2 (13,14,31). However, it is clear that IET in NaR is gated by large conformational changes of the Cyt b domain relative to the electrondonating and nitrate-reducing modules of the enzyme, which is illustrated in Scheme 1, where the gating effects of the two hinges are grouped together.…”

Sulfite-oxidizing enzymes

“…the equilibrium constant, as calculated from the transient trace, see below). Extensive flash photolysis studies on native chicken SO and wild type recombinant human SO have shown that the first-order IET rate constants between the reduced Fe(II) and oxidized Mo(VI) centers depend on the following experimental conditions: solution viscosity [34,77], sulfate concentrations [78][79][80], and pH [34,78,79]; this information is important for understanding the IET mechanism in the enzymatic catalysis (see below).…”