Photoinduced electron-transfer kinetics of singly labeled ruthenium bis(bipyridine) dicarboxybipyridine cytochrome c derivatives

Abstract

Cytochrome c derivatives labeled at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine [RuII(bpy)2(dcbpy)] were prepared by using the procedure described previously [Pan, L. P., Durham, B., Wolinska, J., & Millett, F. (1988) Biochemistry 27, 7180-7184]. Four additional singly labeled derivatives were purified, bringing the total number to 10. These derivatives have a strong luminescence emission centered at 662 nm arising from the excited state, RuII*. Transient absorption spectroscopy was used to directly measure the rate constants for the photoinduced electron-transfer reaction from RuII* to the ferric heme group (k1) and for the thermal back-reaction from the ferrous heme group to RuIII (k2). The rate constants were found to be k1 = 14 X 10(6) s-1 and k2 = 24 X 10(6) s-1 for the derivative modified at lysine 72, which has a distance of 8-16 A between the ruthenium and heme groups. Similar rate constants were found for the derivatives modified at lysines 13 and 27, which have distances of 6-12 A separating the ruthenium and heme groups. The rate constants were significantly slower for the derivatives modified at lysine 25 (k1 = 1 X 10(6) s-1, k2 = 1.5 X 10(6) s-1) and lysine 7 (k1 = 0.3 X 10(6) s-1, k2 = 0.5 X 10(6) s-1), which have distances of 9-16 A. Transients due to photoinduced electron transfer could not be detected for the remaining derivatives, which have larger distances between the ruthenium and heme groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Definition of the Interaction Domain for Cytochrome con the Cytochrome bc 1 Complex

“…This derivative is the best currently available for this application. The rate constant for electron transfer from Ru(II)* to heme c in Ru-72-Cc is k 1 ϭ 3 ϫ 10 7 s Ϫ1 , which is very fast compared with intracomplex electron transfer between heme c and cytochrome c 1 (18). The yield of heme c reduced with a single laser flash is 35%, which is among the largest for the available ruthenium trisbipyridine-labeled cytochrome c deriv- atives.…”

“…In this technique, a 1:1 electrostatic complex between cytochrome bc 1 and a cytochrome c derivative labeled with ruthenium trisbipyridine is formed at low ionic strength. The Ru(II) group is photoexcited to a metalto-ligand charge transfer state, Ru(II)*, which is a strong reducing agent and rapidly transfers an electron to heme c (18). Electron transfer between photoreduced heme c and cytochrome c 1 can be measured on a microsecond time scale (17).…”

Definition of the Interaction Domain for Cytochrome con Cytochrome c Oxidase

“…In this technique, the 1:1 electrostatic complex between CcO and a ruthenium-Cc derivative labeled with ruthenium trisbipyridine is formed at low ionic strength. The Ru(II) group is photoexcited to a metal-toligand charge transfer state, Ru(II*), which is a strong reducing agent and rapidly transfers an electron to heme c (21). The photoreduced heme c in yeast Ru-39-Cc transfers an electron to Cu A in beef CcO with a rate constant of 6 ϫ 10 4 s Ϫ1 , followed by electron transfer from Cu A to heme a with a rate constant of 2 ϫ 10 4 s Ϫ1 (20).…”

Design of photoactive ruthenium complexes to study electron transfer and proton pumping in cytochrome oxidase

“…These investigations were aimed at developing a better understanding of electron transfer within a metalloprotein. Subsequent work in our laboratory led to the preparation of a series of cytochrome c derivatives that contained photoactive Ru(bpy) 2 (bpyCOOH) covalently linked to surface lysines [27,28,29]. Derivatives based on coordinating Ru(bpy) 2 (imidazole) to surface histidines were also developed [30,31].…”