Synthesis and room temperature photo-induced electron transfer in biologically active bis(terpyridine)ruthenium(II)–cytochrome c bioconjugates and the effect of solvents on the bioconjugation of cytochrome c

Photo-active bis(terpyridine)ruthenium(II) chromophores were synthesised and attached to the redox enzyme iso-1 cytochrome c in a mixed solvent system to form photo-induced bioconjugates in greater than 40% yield after purification. The effects of up to 20% (v/v) of acetonitrile, tetrahydrofuran, dimethylformamide, or dimethyl sulfoxide at 4, 25 and 35 °C on the stability and biological activity of cytochrome c and its reactivity towards the model compound 4,4′-dithiodipyridine (DTDP) was measured. The second-order rate constant for the DTDP reaction was found to range between k = 2.5–4.3 M⁻¹ s⁻¹ for reactions with 5% organic solvent added compared to k = 5.6 M⁻¹ s⁻¹ in pure water at 25 °C. Use of 20% solvent generally results in significant protein oxidation, and 20% acetonitrile and tetrahydrofuran in particular result in significant protein dimerization, which competes with the bioconjugation reaction. Cyclic voltammetry studies indicated that the rate of electron transfer to the heme in solution was reduced in the bis(terpyridine)ruthenium(II) cytochrome c bioconjugates compared to unmodified cytochrome c. Steady-state fluorescence studies on these bioconjugates showed that energy or electron transfer is taking place between the bis(terpyridine)ruthenium(II) chromophores and cytochrome c. The bis(terpyridine)ruthenium(II) cytochrome c bioconjugates demonstrate room temperature photo-activated electron transfer from the bis(terpyridine)ruthenium(II) donor to the protein acceptor. Two sacrificial donors were used; in 50% glycerol, the bioconjugates were reduced in about 15 min while in 20 mM EDTA the bioconjugates were fully reduced in less than 5 min upon irradiation with a xenon lamp source. Under these conditions, the reduction of the non-covalent mixture of cytochrome c and bis(terpyridine)ruthenium(II) mixtures took over 30 min. Control experiments showed that the photo-induced reduction of cytochrome c only occurs in the absence of oxygen and presence of a sacrificial donor. These results are encouraging for future incorporation of these bioconjugates in light-responsive bioelectronic circuits, including photo-activated biosensors and biofuel cells.