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 (k_ese) for recombinant Clostridium pasteurianum rubredoxin in the Fe^II and Fe^III forms, referred to here as Rd_red and Rd_ox, has been determined. Using NMR spectroscopy the procedure involves monitoring the perturbation introduced by increasing concentrations of the Rd_ox form on the longitudinal and transverse relaxation times of hyperfine-shifted ¹H NMR signals of Rd_red. A second-order rate constant k_ese(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 k_ese 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 Å 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 pK_a 6.2 for Rd_red and 5.8 for Rd_ox. Rate constants from cross-reaction studies involving the oxidation of Rd_red 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 k_ese for the Rd_red–Rd_ox 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.