Cation-Promoted Cyclic Voltammetry of Recombinant Rat Outer Mitochondrial Membrane Cytochrome b5 at a Gold Electrode Modified with β-Mercaptopropionic Acid

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Reversible cyclic voltammetry of recombinant rat outer membrane (OM) cytochrome b5 was observed at a gold electrode modified with β-mercaptopropionic acid. Electron transfer between the negative electrode surface and the negatively charged OM cytochrome b5 was promoted by the addition of divalent metal ions such as Mg2+ or Ca2+ and by the positively charged species poly-l-lysine. The titration of OM cytochrome b5 (0.1 mM) with poly-l-lysine resulted in a gradual positive shift of the E1/2 value which leveled off at +8 mV vs NHE when the poly-l-lysine:cytochrom b5 ratio reached a value of 2:1. Since the further addition of poly-l-lysine had no effect on the E1/2 value of the protein, it was concluded that a complex is formed in which two molecules of poly-l-lysine bind to each molecule of OM cytochrome b5. When the OM cytochrome b5-poly-l-lysine complex (0.1 mM) was titrated with Mg2+ or Ca2+ ions, the E1/2 value shifted gradually in the negative 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 b5 was promoted by Mg2+ or Ca2+ ions, the minimum concentration of divalent cation necessary to produce a reversible voltammogram was 40 mM and the observed E1/2 was −46 mV vs NHE. On the other hand, only 0.2 mM [Cr(NH3)6]3+ was necessary to promote the reversible electrochemistry of 0.1 mM cytochrome b5. 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 b5 on the kind and concentration of multivalent ions in solution. A reduction potential of −102 mV vs NHE was obtained for OM cytochrome b5 (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, μ = 0.1 M. This value is approximately 100 mV more negative than the reduction potentials reported for microsomal cytochromes b5 obtained from other sources under the same conditions. The binding interactions between OM cytochrome b5 and poly-l-lysine or Mg2+ ions were probed by investigating the isotropically shifted 1H NMR resonances arising from the heme in the OM cytochrome b5. The NMR spectroscopic studies showed that the Mg2+ ions are chelated by the heme propionate in position f, namely, 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 surface of the protein. Poly-l-lysine was found to interact only with the cation binding sites on the surface of the OM cytochrome b5. The differences in reduction potential that were observed for the OM cytochrome b5 in the presence of MgCl2 or poly-l-lysine are believed to be due to modification of surface charge near the heme brought about by the binding of MgCl2 or poly-l-lysine. The results indicate that the large density of positive charge introduced by the binding of two molecules 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 reverse the negative charge on the surface of the OM cytochrome b5. These results also suggest that the reduction potential of OM cytochrome b5 may be modulated by physiological concentrations of Ca2+ or Mg2+ ions and by complex formation with complementarily charged physiological partner proteins. © 1994, American Chemical Society. All rights reserved.

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