Directional electron transfer in ruthenium-modified horse heart cytochrome c

Abstract

Cytochrome c can be modified by [(NH₃)₅Ru^II/III-] specifically at the imidazole moiety of histidine 33, and we have recently discussed the thermodynamics and kinetics of electron transfer within this modified protein^1–5. X-ray crystal structures of the oxidized and reduced forms of tuna cytochrome c⁶ indicate that the separation between the haem group of cytochrome c and the ruthenium label is 12–16 Å. Internal electron transfer from the [(NH₃)₅Ru^II-] centre to the Fe(III) haem centre occurs with a rate constant K ≃ 53s⁻¹ (25 °C) (ΔH^‡=3.5kcal mol⁻¹, ΔS^‡ = −39EU), as measured by pulse radiolysis. The measured unimolecular rate constant¹, k ≃ 53 s⁻¹, is on the same timescale as a number of conformational changes that occur within the cytochrome c molecule^7–9. These results raise the question of whether electron transfer or protein conformational change is the rate limiting step in this process. We describe here an experiment that probes this intramolecular electron transfer step further. It involves reversing the direction of electron transfer by changing the redox potential of the ruthenium label. Electron transfer in the new ruthenium–cytochrome c derivative described here is from haem(II) to the RU(III) label, whereas in (NH₃)₅Ru–cytochrome c the electron transfer is from RU(II) to haem(III). Intramolecular electron transfer from haem(II) to RU(III) in the new ruthenium–cytochrome c described here proceeds much slower (> 10⁵ times) than the electron transfer from Ru(II) to haem(III) in the (NH₃)₅Ru–cytochrome c. We therefore conclude that electron transfer in cytochrome c is directional, with the protein envelope presumably involved in this directionality.