Preparation, characterisation, and intramolecular electron-transfer studies on the penta-ammineruthenium histidine-modified high-potential iron–sulphur protein from Chromatium vinosum

Abstract

The high-potential iron–sulphur protein (Hipip) from Chromatium vinosum(Mrca. 9 500; 85 amino acids) contains a single histidine residue, His 42, the location of which is known from the X-ray crystal structure. The reaction of Hipipred with excess [Ru(NH3)5(H2O)]2+ followed by oxidation, yields the Ru-modified protein Hipipox·RuIII. The product has been purified by column chromatography, and analysed by inductively coupled plasma atomic emission spectroscopy (Fe : Ru ratio 4 : 1). The His 42 of the Ru-modified protein no longer reacts with diethyl pyrocarbonate (depc), and the sharp 1H n.m.r. C2H resonance of His 42 at 8.3 p.p.m. is no longer present due to paramagnetic line broadening by the attached RuIII. The reduction potential of the [Fe4S4]3+/2+ couple remains at 350 mV in the RuIII-modified protein. On pulse radiolysis using eaq–(CO2˙– or O2–) as reductant to generate Hipipox·RuII, a second stage assigned to intramolecualr formation of Hipipred·RuIII is observed, k= 18 s–1(pH 7.0, 20 °C). An additional step is observed as a result of autoreduction of Hipipox·RuIII to Hipipred·RuIII, which occurs on bubbling Ar gas through solutions in anaerobic procedures. Subsequent pulsing generates some Hipipred·RuII, the decay of which gives rise to third stage here assigned as an intermolecular electron transfer between fully oxidised and fully reduced Ru-modified protein. The His 42 is near to the Fe4S4 active site, with Cys 43 attached to one of the Fe atoms. The direct donor–acceptor linkage is unusually short and the intramolecular rate constant is therefore of interest in the context of through-space (7.9 A) or through-bond (13 A) electron transfer. There is no obvious enhancement of the intramolecular electron-transfer process, and at this time no reason for favouring the through-bond assignment.