Preparation and intramolecular electron-transfer rate constant for the ruthenium-modified selenium-substituted [4Fe–4Se] high-potential protein from Chromatium vinosum and related studies

Abstract

The Se-substituted [4Fe–4Se] form of the high-potential iron–sulfur protein from Chromatium vinosum(M_r≈9500, 85 amino acids) has been prepared and Ru-modified (68% yield) at the single histidine residue His 42. The purified product gives and Fe: Ru ratio close to 4:1 consistent with a single stoichiometric modification. The His 42 of the Ru-modified protein no longer reacts with O(CO₂Et)₂ and the sharp ¹H NMR C₂H resonance for His 42 at δ 8.38 is no longer present due to paramagnetic line broadening by the attached Ru^III. The His 42 is bonded via Cys 43 to the active site, with an edge-to-edge distance close to 7.9 Å. Pulse-radiolysis reduction of the fully oxidised Fe₄Se₄³⁺–Ru^III protein with e_aq^– gives the metastable product Fe₄Se³⁺–Ru^II. A second slower stage, corresponding to conversion of the Fe₄Se₄³⁺–Ru^II to Fe₄Se₄²⁺–Ru^III is made up of intramolecular (1.30 s^–1) and intermolecular (2.65 × 10⁵ M^–1 s^–1) electron-transfer steps. The k_intra step is of interest alongside the earlier value (18 s^–1) for native [4Fe–4S] protein. The smaller reduction potential for the Fe₄Se₄^3+/2+(285 mV) as compared to Fe₄S₄^3+/2+(350 mV) couple is believed to be the major contributing factor to the slower intramolecular electron-transfer process. The Beratan–Onuchic pathways program has been used and indicates through-bond electron transfer from the Ru at His 42 to the cluster as the most favourable route. The rate constant at 25 °C (M^–1 s^–1) for the [Fe(CN)₆]^3– oxidation of the Fe₄Se₄²⁺ protein has also been determined (4.5 × 10³ M^–1 s^–1), and is less than that for native Fe₄S₄²⁺ protein (2.0 × 10³ M^–1 s^–1) at pH 7.0, /= 0.100 M (NaCl).