The reactions of bovine cytochrome c oxidase with horse cytochrome c derivatives labeled at specific lysine amino groups with (dicarboxybipyridine)bis(bipyridine)ruthenium (II) were studied by laser flash photolysis. All of the derivatives form complexes with cytochrome c oxidase at low ionic strength (5 mM sodium phosphate, pH 7). Excitation of Ru(II) to Ru(II*) with a short laser flash resulted in rapid electron transfer to the ferric heme group of cytochrome c, followed by electron transfer to cytochrome c oxidase. The photoreduced heme Fe(II) in the cytochrome c derivative modified at lysine 25 on the periphery of the heme crevice domain transferred an electron to CuA with a rate constant of 1.1 x 10(4) s-1. CuA then transferred an electron to cytochrome a with a rate constant of 2.3 x 10(4) s-1. The derivatives modified at lysines 7, 39, 55, and 60 remote from the heme crevice domain of cytochrome c have nearly the same kinetics. The rate constant for electron transfer from the cytochrome c heme to CuA is greater than 10(5) s-1, and the rate constant for electron transfer from CuA to cytochrome a is 2 x 10(4) s-1. The cytochrome c derivatives modified at lysines 13 and 27 in the heme crevice domain react much more slowly than the other derivatives, with intracomplex rate constants for oxidation of cytochrome c ranging from 1000 to 6000 s-1. The bulky ruthenium group at the heme crevice domain of these derivatives apparently alters the binding orientation, leading to smaller electron-transfer rates.(ABSTRACT TRUNCATED AT 250 WORDS)
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)
A novel two-step procedure has been developed to prepare cytochrome c derivatives labeled at specific lysine amino groups with ruthenium bis(bipyridine) dicarboxybipyridine [RuII(bpy)2(dcbpy)]. In the first step, cytochrome c was treated with the mono-N-hydroxysuccinimide ester of 4,4'-dicarboxy-2,2'-bipyridine (dcbpy) to convert positively charged lysine amino groups to negatively charged dcbpy-lysine groups. Singly labeled dcbpy-cytochrome c derivatives were then separated and purified by ion-exchange chromatography. In the second step, the individual dcbpy-cytochrome c derivatives were treated with RuII(bpy)2CO3 to form singly labeled RuII(bpy)2(dcbpy-cytochrome c) derivatives. The specific lysine labeled in each derivative was determined by reverse-phase chromatography of a tryptic digest. All of the derivatives had a strong luminescence emission centered at 662 nm, but the luminescence decay rates were increased relative to that of a non-heme protein control, RuII(bpy)2(dcbpy-lysozyme), which was 1.8 X 10(6) s-1. The luminescence decay rates were found to be 21, 16, 7.2, 5.7, 4.3, 4.3, and 3.5 X 10(6) s-1 for derivatives singly labeled at lysines 13, 72, 25, 7, 39, 86, and 87, respectively. There was an inverse relationship between the luminescence decay rates and the distances between the ruthenium labels and the heme group. The increased luminescence decay rates observed in the cytochrome c derivatives might be due to electron transfer from the excited triplet state of ruthenium to the ferric heme group. However, it is also possible that an energy-transfer mechanism might contribute to the luminescence quenching.(ABSTRACT TRUNCATED AT 250 WORDS)
A new technique has been developed to measure intracomplex electron transfer between cytochrome c and its redox partners. Cytochrome c derivatives labeled at single lysine amino groups with ruthenium bisbipyridine dicarboxybipyridine were prepared as previously described [Pan, L.P., Durham, B., Wolinska, J., & Millett, F. (1988) Biochemistry 27, 7180-7184]. Excitation of RuII with a short light pulse resulted in the formation of the excited-state RuII*, which rapidly transferred an electron to the ferric heme group to form FeII and RuIII. Aniline was included in the buffer to reduce RuIII to RuII, leaving the heme group in the ferrous state. This process was complete within the lifetime of the light pulse. When plastocyanin was present in the solution, electron transfer from the ferrous heme of cytochrome c to CuII in plastocyanin was observed. All of the ruthenium cytochrome c derivatives formed electrostatic complexes with plastocyanin at low ionic strength, allowing intracomplex electron-transfer rate constants to be measured. The rate constants for derivatives modified at the indicated lysines were as follows: Lys 13, 1920 s-1; Lys 8, 1480 s-1; Lys 7, 1340 s-1; Lys 86, 1020 s-1; Lys 25, 820 s-1; Lys 72, 800 s-1; Lys 27, 530 s-1. It is interesting that the derivative modified at lysine 13 at the top of the heme crevice had the largest rate constant, while lysine 27 at the right side of the heme crevice had the smallest.(ABSTRACT TRUNCATED AT 250 WORDS)
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