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(Mr≈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 RuIII. 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+–RuIII protein with eaq– gives the metastable product Fe4Se3+–RuII. A second slower stage, corresponding to conversion of the Fe4Se43+–RuII to Fe4Se42+–RuIII is made up of intramolecular (1.30 s–1) and intermolecular (2.65 × 105 M–1 s–1) electron-transfer steps. The kintra step is of interest alongside the earlier value (18 s–1) for native [4Fe–4S] protein. The smaller reduction potential for the Fe4Se43+/2+(285 mV) as compared to Fe4S43+/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).