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

Abstract

This review describes the development and application of photoactive ruthenium complexes to study electron transfer and proton pumping reactions in cytochrome c oxidase (CcO). CcO uses four electrons from Cc to reduce O2 to two waters, and pumps four protons across the membrane. The electron transfer reactions in cytochrome oxidase are very rapid, and cannot be resolved by stopped-flow mixing techniques. Methods have been developed to covalently attach a photoactive tris(bipyridine)ruthenium group [Ru(II)] to Cc to form Ru-39-Cc. Photoexcitation of Ru(II) to the excited state Ru(II*), a strong reductant, leads to rapid electron transfer to the ferric heme group in Cc, followed by electron transfer to CuA in CcO with a rate constant of 60,000 s−1. Ruthenium kinetics and mutagenesis studies have been used to define the domain for the interaction between Cc and CcO. New ruthenium dimers have also been developed to rapidly inject electrons into CuA of CcO with yields as high as 60%, allowing measurement of the kinetics of electron transfer and proton release at each step in the oxygen reduction mechanism.

Light-Triggered Modulation of Cellular Electrical Activity by Ruthenium Diimine Nanoswitches

“…The excitation and emission spectra, as well as the structure of RubpyC17 are shown in Figure 1A. RubpyC17 applied to the bath at a final concentration of 10 μM rapidly and stably incorporates into mammalian cell membranes as shown by plasma membranelocalized luminescence 30,31 ( Figure 1B Figure 1B, bottom). None of these cell types exhibited significant autofluorescence or autoluminescence in the red channel, as shown by lack of emission collected from cells not exposed to RubpyC17.…”

Ru(II)-diimine functionalized metalloproteins: From electron transfer studies to light-driven biocatalysis

“…Millet, Durham and coworkers have used a cascade of electron transfer steps initiated at the ruthenium modified cytochrome c to eventually promote the reduction of cytochrome c oxidase. [19, 20, 47] This approach has also enabled the study of proton pumping across the membrane accompanying the oxygen reduction. [19]…”

“…[19, 20, 47] This approach has also enabled the study of proton pumping across the membrane accompanying the oxygen reduction. [19]…”

“…The following sections will introduce the various synthetic strategies to covalently attach these complexes to proteins and the techniques to characterize their attachment. Then, the significant contributions and conclusions from the extensive studies on electron transfer in metalloproteins conducted by the Gray group [7-10] and others [19-21] will be highlighted. These studies have paved the way for the detection of elusive reaction intermediates in heme proteins and eventually to the development of light-driven biocatalysis.…”

Dynamics of physical processes in proteins studied by kinetic absorption spectroscopy

“…The modification of the metal coordinating ligands allows the tuning of the midpoint redox potential. A number of compounds with redox potential in the range from -0.87 to -0.37 V have been synthesized (Millett and Durham, 2002;Engstrom et al, 2003;Geren et al, 2009;Durham and Millett, 2012), and after covalent or electrostatic attachment, successfully applied as electron donors to different redox centers within the protein matrix, including heme (myoglobin (Cowan et al, 1988), cytochrome c, cytochrome c oxidase (Durham and Millett, 2012), blue copper (Bechtold et al, 1986) and iron-sulfur sites (Sola et al, 1989)). To monitor the events Introduction which follow the initiation of electron transfer, a range of spectroscopic methods can be employed.…”