Electron self-exchange and cross-reaction studies on wild-type Clostridium pasteurianum rubredoxin and its val-8→Glu variant

Abstract

The electron self-exchange rate constant (kese) for recombinant Clostridium pasteurianum rubredoxin in the FeII and FeIII forms, referred to here as Rdred and Rdox, has been determined. Using NMR spectroscopy the procedure involves monitoring the perturbation introduced by increasing concentrations of the Rdox form on the longitudinal and transverse relaxation times of hyperfine-shifted 1H NMR signals of Rdred. A second-order rate constant kese(25 °C) of 1.6 × 105 M–1 s–1 has been obtained at pH 6.5, I= 0.100 M (NaCl). Similar measurements carried out with the molecular variant in which valine-8 is replaced by glutamate (Val8Glu) give a significantly smaller kese value of 4.7 × 103 M–1 s–1. The effect of the negatively charged Glu-8, adjacent to the surface-exposed Cys-9 and Cys-42 residues of the Fe active site, suggests a close Fe–Fe approach of ≈12 A for electron exchange. The reduction potential vs. NHE of rubredoxin by cyclic and square-wave voltammetry (using a promotor) determined as –81 mV is pH invariant, but that of Val8Glu (– 73 mV at pH 6.5) depends on pH, with pKa 6.2 for Rdred and 5.8 for Rdox. Rate constants from cross-reaction studies involving the oxidation of Rdred by Pseudomonas aeruginosa azurin and cytochrome c551 have also been determined using the stopped-flow method, and kinetic data analysed in the framework of the Marcus theory. Azurin in particular with its close to electroneutral surface has been successfully used as a redox partner in other interprotein cross-reaction studies. However calculated electron self-exchange rate constants kese for the Rdred–Rdox couple average 35 M–1 s–1, and are > 103 smaller than the value determined by NMR spectroscopy, indicating the possible involvement of a different reaction site for these reactions.