CATION-PROMOTED CYCLIC VOLTAMMETRY OF RECOMBINANT RAT OUTER MITOCHONDRIAL-MEMBRANE CYTOCHROME B(5) AT A GOLD ELECTRODE MODIFIED WITH BETA-MERCAPTOPROPIONIC ACID

Reversible cyclic voltammetry of recombinant rat outer membrane (OM) c ytochrome b(5) was observed at a gold electrode modified with beta-mer captopropionic acid. Electron transfer between the negative electrode surface and the negatively charged OM cytochrome b(5) was promoted by the addition of divalent metal ions such as Mg2+ or Ca2+ and by the po sitively charged species poly-L-lysine. The titration of OM cytochrome b(5) (0.1 mM) with poly-L-lysine resulted in a gradual positive shift of the E(1/2) value which leveled off at +8 mV vs NHE when the poly-L -lysine:cytochrome b(5) ratio reached a value of 2:1. Since the furthe r addition of poly-L-lysine had no effect on the E(1/2) value of the p rotein, it was concluded that a complex is formed in which two molecul es of poly-L-lysine bind to each molecule of OM cytochrome b(5). When the OM cytochrome b(5)-poly-L-lysine complex (0.1 mM) was titrated wit h Mg2+ or Ca2+ ions, the E(1/2) value shifted gradually in the negativ e direction and leveled off at -40 mV vs NHE when the concentration of divalent ions reached 85 mM. When the voltammetric response of 0.1 mM cytochrome b(5) was promoted by Mg2+ or Ca2+ ions, the minimum concen tration of divalent cation necessary to produce a reversible voltammog ram was 40 mM and the observed E(1/2) was -46 mV vs NHE. On the other hand, only 0.2 mM [Cr(NH3)(6)](3+) was necessary to promote the revers ible electrochemistry of 0.1 mM cytochrome bg The half-wave potential observed under these conditions was -78 mV vs NHE. This indicates that there is a large dependence of the reduction potential of cytochromes b(5) on the kind and concentration of multivalent ions in solution. A reduction potential of -102 mV vs NHE was obtained for OM cytochrome b(5) (0.60 mM) by spectroelectrochemical titration in the presence of 0.4 mM [RU(NH3)(6)](3+) and 1 mM methyl viologen, pH 7.0, mu = 0.1 M. This value is approximately 100 mV more negative than the reduction po tentials reported for microsomal cytochromes b(5) obtained from other sources under the same conditions. The binding interactions between OM cytochrome b(5) and poly-L-lysine or Mg2+ ions were probed by investi gating the isotropically shifted H-1 NMR resonances arising from the h eme in the OM cytochrome b(5). The NMR spectroscopic studies showed th at the Mg2+ ions are chelated by the heme propionate in position f, na mely, heme propionate 6 in isomer A and heme propionate 7 in isomer B, and by the carboxylate groups of the cation binding sites on the surf ace of the protein. Poly-L-lysine was found to interact only with the cation binding sites on the surface of the OM cytochrome b(5). The dif ferences in reduction potential that were observed for the OM cytochro me b(5) in the presence of MgCl2 or poly-L;lysine are believed to be d ue to modification of surface charge near the heme brought about by th e binding of MgCl2 or poly-L-lysine. The results indicate that the lar ge density of positive charge introduced by the binding of two molecul es of poly-L-lysine results in a positive shift of reduction potential , while the binding of Mg2+ to the surface of the protein does not rev erse the negative charge on the surface of the OM cytochrome b(5) Thes e results also suggest that the reduction potential of OM cytochrome b (5) may be modulated by physiological concentrations of Ca2+ or Mg2+ i ons and by complex formation with complementarily charged physiologica l partner proteins.