Design of a Ruthenium-Labeled Cytochrome c Derivative to Study Electron Transfer with the Cytochrome bc₁ Complex

Abstract

A new ruthenium-cytochrome c derivative was designed to study electron transfer from cytochrome bc1 to cytochrome c (Cc). The single sulfhydryl on yeast H39C;C102T iso-1-Cc was labeled with Ru(2,2'-bipyrazine)2(4-bromomethyl-4'-methyl-2,2'-bipyridine) to form Ru(z)-39-Cc. The Ru(z)-39-Cc derivative has the same steady-state activity with yeast cytochrome bc1 as wild-type yeast iso-1-Cc, indicating that the ruthenium complex does not interfere in the binding interaction. Laser excitation of reduced Ru(z)-39-Cc results in electron transfer from heme c to the excited state of ruthenium with a rate constant of 1.5 x 10(6) x s(-1). The resulting Ru(I) is rapidly oxidized by atmospheric oxygen in the buffer. The yield of photooxidized heme c is 20% in a single flash. Flash photolysis of a 1:1 complex between reduced yeast cytochrome bc1 and Ru(z)-39-Cc at low ionic strength leads to rapid photooxidation of heme c, followed by intracomplex electron transfer from cytochrome c1 to heme c with a rate constant of 1.4 x 10(4) x s(-1). As the ionic strength is raised above 100 mM, the intracomplex phase disappears, and a new phase appears due to the bimolecular reaction between solution Ru-39-Cc and cytochrome bc1. The interaction of yeast Ru-39-Cc with yeast cytochrome bc1 is stronger than that of horse Ru-39-Cc with bovine cytochrome bc1, suggesting that nonpolar interactions are stronger in the yeast system.

Electronic Connection Between the Quinone and CytochromecRedox Pools and Its Role in Regulation of Mitochondrial Electron Transport and Redox Signaling

“…Electrostatic forces are considered to be a dominant factor that contributes to binding of cytochrome c to cytochrome bc 1 , which is inferred from a significant salt dependence of electron transfer between these proteins (90,114,121,135,249). An importance of short-range hydrophobic interactions between surfaces of cytochrome c 1 subunit and cytochrome c was proposed on the basis of X-ray structures of cytochrome bc 1 co-crystallized with its redox partner (152,238).…”

Chapter 5 Use of Ruthenium Photooxidation Techniques to Study Electron Transfer in the Cytochrome bc1 Complex

“…The new brominated Ru(bpz) 2 (dmb) reagent can be used to covalently attach a ruthenium complex to a protein containing a single cysteine sulfhydryl group on its surface (Engstrom et al, 2003). In order to label yeast Cc, the single Cys-102 in wild-type yeast iso-1-Cc is first mutated to Thr, and then His-39 is mutated to Cys to form H39C;C102T yeast iso-1-Cc.…”

“…In one method, a polypyridyl ruthenium complex [Ru(II)] is covalently attached to cytochrome c to form Ru-Cc (Engstrom et al, 2003). Photoexcitation of Ru(II) to the metal-to-ligand charge-transfer state, Ru(II*), a strong oxidant, leads to rapid oxidation of the ferrous heme group in Cc.…”

“…Subsequent electron transfer from cytochrome c 1 to the Ru-Cc heme can be measured on a time scale as fast as 50 nanoseconds (Scheme 1). This new technique has been used to measure intracomplex electron transfer between cytochrome bc 1 and Cc for the first time (Engstrom et al, 2003). In a second method, a new ruthenium dimer has been developed which binds with high affinity to cytochrome bc 1 and can photooxidize cyt c 1 within 1 μs (Sadoski et al, 2000).…”

Molecular Organization of Cytochrome c2 near the Binding Domain of Cytochrome bc1 Studied by Electron Spin–Lattice Relaxation Enhancement

“…Increasing salt concentration or mutations of some charged residues resulted in the progressive disappearance of the complexes observed by EPR 14 or in a change of the ET kinetics from intermolecular to bimolecular. 11 These observations were interpreted in terms of a collisional model of ET between cytochrome c 2 and cytochrome bc 1 . 14 …”