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(M_rca. 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 Hipip_red with excess [Ru(NH₃)₅(H₂O)]²⁺ followed by oxidation, yields the Ru-modified protein Hipip_ox·Ru^III. 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 Ru^III. The reduction potential of the [Fe₄S₄]^3+/2+ couple remains at 350 mV in the Ru^III-modified protein. On pulse radiolysis using e_aq^–(CO₂˙^– or O₂^–) as reductant to generate Hipip_ox·Ru^II, a second stage assigned to intramolecualr formation of Hipip_red·Ru^III is observed, k= 18 s^–1(pH 7.0, 20 °C). An additional step is observed as a result of autoreduction of Hipip_ox·Ru^III to Hipip_red·Ru^III, which occurs on bubbling Ar gas through solutions in anaerobic procedures. Subsequent pulsing generates some Hipip_red·Ru^II, 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 Å) or through-bond (13 Å) 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.