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 μs 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 × 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 μs 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 × 10 4 s -1 in R. sphaeroides cyt bc 1 using 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.

Roles of DMSO-type ruthenium complexes in disaggregation of prion neuropeptide PrP106–126

“…[76][77][78] In fact, these complexes also interact with proteins, enzymes, or polypeptide fragments. 2,79,80 The results of our ESI-MS experiments showed that metal coordination predominated the binding interaction; this nding is consistent with that of a previous report. 10 Moreover, the decrease in CV peak current, proton relaxation in NMR, and K d determination suggested that hydrophobic interaction existed between the peptide and Ru complexes.…”

An electron transfer path connects subunits of a mycobacterial respiratory supercomplex

“…In particular, the carbonyl groups of ubiquinone are coordinated by the conserved His and Asp amongst bc 1 complexes; these residues are substituted by QcrB Trp 231 and QcrB Ser 261 in the bcc-type CIII of M. smegmatis. The carbonyl groups of MK in SC III-IV interact with the side chains of QcrB Tyr 48 and QcrB Ser 261 that may supply protons for MK reduction. The MK head group is ideally placed for electron transfer, being within 5 Å of the A-edge of heme b H .…”

“…The cluster domain undergoes a "head displacement" conformation change to the c 1 -state, in which the [2Fe-2S] cluster moves to within 11 Å of the c 1 heme to facilitate electron transfer. This head displacement or "gating" step occurs with a rate constant of 6 × 10 4 s -1 (48).…”

Recent advances in cytochrome bc1: Inter monomer electronic communication?

“…Second, inter monomer electronic communication per se deserves further studies. Direct determinations of inter monomer hemes b electron transfer rates might be achieved using photo‐activatable ruthenium cytochrome c derivatives [35]. The role of aromatic residues, like F195 and Y199 of cytochrome b , located between the hemes b L at the interface of the monomers in the dimeric cytochrome bc 1 with respect to inter monomer electron transfer [36,37] need reexamination.…”