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 Clos tridium pasteurianum rubredoxin in the Fe-II and Fe-III forms, referre d to here as Rd(red) and Rd(ox), has been determined. Using NMR spectr oscopy the procedure involves monitoring the perturbation introduced b y increasing concentrations of the Rd(ox) form on the longitudinal and transverse relaxation times of hyperfine-shifted H-1 NMR signals of R d(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). Simil ar measurements carried out with the molecular variant in which valine -8 is replaced by glutamate (Val8Glu) give a significantly smaller k(e se) value of 4.7 x 10(3) M(-1) s(-1). The effect of the negatively cha rged 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 in volving the oxidation of Rd(red) by Pseudomonas aeruginosa azurin and cytochrome c(551) have also been determined using the stopped-flow met hod, 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-reac tion 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, indicat ing the possible involvement of a different reaction site for these re actions.