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

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 H-1 NMR signals of Rd(red). A second-order rate constant k(ese) (25 degrees C) of 1.6 x 10(5) 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 x 10(3) 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 approximate to 12 Angstrom for electron exchange. The reduction potential us. 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 greater than or equal to 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 c(551) 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 > 10(3) smaller than the value determined by NMR spectroscopy, indicating the possible involvement of a different reaction site for these reactions.