Chapter 5 Use of ruthenium photooxidation techniques to study electron transfer in the cytochrome bc1 complex

Abstract

Ruthenium photooxidation methods are presented to study electron transfer between the cytochrome bc(1) complex and cytochrome c and within the cytochrome bc(1) complex. Methods are described to prepare a ruthenium cytochrome c derivative, Ru(z)-39-Cc, by labeling the single sulfhydryl on yeast H39C;C102T iso-1-Cc with the reagent Ru(bpz)(2)(4-bromomethyl-4'-methylbipyridine). The ruthenium complex attached to Cys-39 on the opposite side of Cc from the heme crevice does not affect the interaction with cyt bc(1). Laser excitation of reduced Ru(z)-39-Cc results in photooxidation of heme c within 1 microsec with a yield of 20%. Flash photolysis of a 1:1 complex between reduced yeast cytochrome bc(1) and Ru(z)-39-Cc leads to electron transfer from heme c(1) to heme c with a rate constant of 1.4 x 10(4) s(-1). Methods are described for the use of the ruthenium dimer, Ru(2)D, to photooxidize cyt c(1) in the cytochrome bc(1) complex within 1 microsec with a yield of 20%. Electron transfer from the Rieske iron-sulfur center [2Fe2S] to cyt c(1) was detected with a rate constant of 6 x 10(4) s(-1) in R. sphaeroides cyt bc(1) with this method. This electron transfer step is rate-limited by the rotation of the Rieske iron-sulfur protein in a conformational gating mechanism. This method provides critical information on the dynamics of rotation of the iron-sulfur protein (ISP) as it transfers electrons from QH(2) in the Q(o) site to cyt c(1). These ruthenium photooxidation methods can be used to measure many of the electron transfer reactions in cytochrome bc(1) complexes from any source.

Figure 1

Photoinduced electron transfer within yeast Ru_z-39-Cc. A: A solution of reduced 5 μM Ru_z-39-Cc in 5 mM sodium phosphate, pH 7.0 was photoexcited with a 356 nm Nd-Yag laser flash, and the absorbance at 434 nm and 550 nm was monitored. B: The same sample as in A at a longer time scale in the presence and absence of atmospheric oxygen.

Figure 2

Photoinduced electron transfer between yeast Ru-39-Cc and yeast cyt bc₁. A solution containing reduced 5.2 μM yeast Ru_z-39-Cc and yeast cyt bc₁ in 5 mM sodium phosphate, pH 7.0, 250 mM NaCl, and 0.1% lauryl maltoside was photoexcited with a 480 nm laser flash. The 550 nm transient shows Cc photooxidation and reduction, while the 557 nm transient shows cyt c₁ oxidation.

Figure 3

Ionic strength dependence of photoinduced electron transfer between yeast Ru_z-39-Cc and yeast cyt bc₁. The reaction was measured under the same conditions as in Figure 3 with 0 to 800 mM NaCl.

Figure 4

Photoinduced electron transfer within wild-type R. sphaeroides cyt bc₁. A sample containing 5 μM cyt bc₁, 20 μM Ru₂D, 5 mM [Co(NH₃)₅Cl]²⁺, in 20 mM sodium borate, pH 9.0 with 0.01% dodecylmaltoside was treated with 10 μM Q_oC₁₀BrH₂, 1 mM succinate, and 50 nM SCR to reduce [2Fe2S] and cyt c₁, and reduce cyt b_H by about 30%. Excitation of Ru₂D with 480 nm laser flash led to photooxidation of cyt c₁ within 1 μs, followed by reduction in a biphasic reaction with rate constants of 60,000 s^-1 and 2,000 s^-1 as shown in the 552 nm transient. The 561 - 569 transient shows reduction of cyt b_H with a rate constant of 2,300 s^-1.

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