Cytochrome electron transfer studies

An important model system for electron transfer studies is the electron carrier cytochrome c (Cyt c). Its redox center is a heme, coordinated by a histidine and... 

Electron-transfer chains in plants differ in several striking aspects from their mammalian counterparts. Plant mitochondria are well known to contain alternative oxidase that couples oxidation of hydroquinones (e.g., ubiquinol) directly to reduction of oxygen. Semiquinones (anion-radicals) and superoxide ions are formed in such reactions. The alternative oxidase thus provides a bypass to the conventional cytochrome electron-transfer pathway and allows plants to respire in the presence of compounds such as cyanides and carbon monoxide. There are a number of studies on this problem (e.g., see Affourtit et al. 2000, references therein). 

E. Mechanistic Studies on P. pantotrophus Cytochrome cdi 1. Electron Transfer Studied by Pulse Radiolysis... 

Some of the first protein systems where pulse radiolysis was used to help determine mechanism were those of blue copper proteins. These are proteins that are blue in solution and contain what are known as type (I) and type (2) copper centers. Two of the most well-known and well-characterized examples of these are azurin and cytochrome c. It was the studies of these systems that opened up the field of long-distance electron transfer in proteins and, by using the protein structure as a framework for electron transfer through space and through bonds, allowed for the development of a broad theoretical basis and many fascinating experiments on long-range electron transfer. Here, I will limit the discussion to electron transfer studies in azurin as illuminated by pulse radiolysis studies. ... 

The introduction and implementation of heteronuclear-based multidimensional techniques have revolutionized the protein NMR field. Large proteins (> 100 residues) are now amenable to detailed NMR studies and structure determination. These techniques, however, necessarily require a scheme by which and isotopes can be incorporated into the protein to yield a uniformly labeled sample. Additional complications, such as extensive covalent post-translational modifications, can seriously limit the ability to efficiently and cost effectively express a protein in isotope enriched media - the c-type cytochromes are an example of such a limitation. In the absence of an effective labeling protocol, one must therefore rely on more traditional proton homonuclear NMR methods. These include two-dimensional (1) and, more recently, three-dimensional H experiments (2,3). Cytochrome c has become a paradigm for protein folding and electron transfer studies because of its stability, solubility and ease of preparation. As a result, several high-resolution X-ray crystal structure models for c-type cytochromes, in both redox states, have emerged. Although only subtle structural differences between redox states have been observed in these... 

Ruthenium complexes are excellent reagents for protein modification and electron-transfer studies. Ru +-aquo complexes readily react with surface His residues on proteins to form stable derivatives [20, 21]. Low-spin pseudo-octahedral Ru-complexes exhibit small structural changes upon redox cycling between the Ru + and Ru + formal oxidation states [3, 22]. Hence, the inner-sphere barriers to electron transfer (Ai) are small. With the appropriate choice of ligand, the Ru + + reduction potential can be varied from <0.0 to >1.5 V versus NHE [23]. Ru-bpy complexes bound to Lys and Cys residues have been employed to great advantage in studies of protein-protein ET reactions. The kinetics of electron transfer in cytochrome 65/cytochrome c [24], cytochrome c/cytochrome c peroxidase [12], and cytochrome c/cytochrome c oxidase [25] complexes have been measured with the aid of laser-initiated ET from a Ru-bpy label. 

More detailed electron transfer studies indicate that cytochrome C55U and the MADH-amicyanin complex associate in solution (186). The temperature dependence of the rate constant for electron transfer from copper to iron, within this ternary complex, provides estimates of A and Hab of 1.1 eV and 0.3 cm" respectively. These results are consistent with electron transfer over distances of 13-23 A, de-... 

The reorganization energies for ET between the dinuclear Cua center and cytochrome a in cytochrome c oxidase have been estimated to be between 0.15-0.5 eV from ET experiments. This estimate means that the A for the Cua center is even lower than that for the mononuclear cupredoxin center. An electron transfer study of an engineered Cua azurin showed that the calculated A for Cua in the engineered azurin is 0.4eV, which is about half that of the blue copper center in native azurin. " Furthermore, an excited-state distortion analysis of a resonance Raman enhancement profile for Cua also confirmed that the inner-sphere A for Cua is about 50% that of a blue copper center., i7,ioi lower A for Cua can be attributed to the valence delocalization of the dinuclear center the bond length distortions associated with the redox reaction can be spread over two metal ions and thus reduced to 1/2 that of the mononuclear copper or valence-trapped dinuclear centers. 

Dio genes ICN, Nart FC, Temperini MLA, Moreira IS. The [Ru(CN)5(pyS)]4- complex, an efficient self-assembled monolayer for the cytochrome c heterogeneous electron transfer studies. Inorg Chem 2001 40 4884-4889. 

A more detailed study of the biological oxidation of sulphoxides to sulphones has been reported165. In this study cytochrome P-450 was obtained in a purified form from rabbit cells and was found to promote the oxidation of a series of sulphoxides to sulphones by NADPH and oxygen (equation 56). Kinetic measurements showed that the process proceeds by a one-electron transfer to the activated enzymatic intermediate [an oxenoid represented by (FeO)3+] according to equation (57). 

Studies (see, e.g., (101)) indicate that photosynthesis originated after the development of respiratory electron transfer pathways (99, 143). The photosynthetic reaction center, in this scenario, would have been created in order to enhance the efficiency of the already existing electron transport chains, that is, by adding a light-driven cycle around the cytochrome be complex. The Rieske protein as the key subunit in cytochrome be complexes would in this picture have contributed the first iron-sulfur center involved in photosynthetic mechanisms (since on the basis of the present data, it seems likely to us that the first photosynthetic RC resembled RCII, i.e., was devoid of iron—sulfur clusters). 

Cyclic voltammetry and other electrochemical methods offer important and sometimes unique approaches to the electroactive species. Protein organization and kinetic approaches (Correia dos Santos et al. 1999, Schlereth 1999) can also be studied by electrochemical survey. The electron transfer reaction between cytochrome P450scc is an important system for... 

C. Shen and N.M. Kostic. Kinetics of photoinduced electron-transfer reactions within sol-gel silica glass doped with zinc cytochrome c. Study of electrostatic effects in confined liquids. J. Am. Chem. Soc. 119, 1304-1312 (1997). 

The first reports on direct electrochemistry of a redox active protein were published in 1977 by Hill [49] and Kuwana [50], They independently reported that cytochrome c (cyt c) exhibited virtually reversible electrochemistry on gold and tin doped indium oxide (ITO) electrodes as revealed by cyclic voltammetry, respectively. Unlike using specific promoters to realize direct electrochemistry of protein in the earlier studies, recently a novel approach that only employed specific modifications of the electrode surface without promoters was developed. From then on, achieving reversible, direct electron transfer between redox proteins and electrodes without using any mediators and promoters had made great accomplishments. 

Z.Q. Feng, S. Imabayashi, T. Kakiuchi, and K. Niki, Electroreflectance spectroscopic study of the electron transfer rate of cytochrome c electrostatically immobilized on the w-carboxyl alkanethiol monolayer modified gold electrode. J. Electroanal. Chem. 394, 149-154 (1995). 

It is timely to review the reactivity of plastocyanin in the light of recent aqueous solution studies, and the elegant structural work of Freeman and colleagues on both the PCu(I) and PCu(II) forms (1 2) Plastocyanin now ranks alongside cytochrome c (3) as the electron-transfer metalloprotein for which there is most structural information. 

Complex I can be regulated by phosphorylation. Demin et al. [28] studied superoxide generation by Complex III using the kinetics model of electron transfer from succinate to cytochrome c. 

Recent advances in measuring the kinetics of the various electron-transfer steps in this system have been achieved by use of flash photolysis of ruthenated derivatives of cytochrome c (Ru-Cc) (17-19). In these studies [Ru(bpy)3]2+ is covalently bound to a surface residue at a site that does not interfere with the docking of cytochrome c to cytochrome c oxidase. Solutions are then prepared containing both Ru-Cc and cytochrome c oxidase, and the two proteins associate to form a 1 1 complex. Flash photolysis of the solution leads directly to the excitation of the RuII(bpy)3 site, which then reduces heme c very rapidly. This method thus provides a convenient means to observe the subsequent intracomplex electron transfer from heme c to cytochrome c oxidase and further stages in the process. 

In the following sections the effect of pressure on different types of electron-transfer processes is discussed systematically. Some of our work in this area was reviewed as part of a special symposium devoted to the complementarity of various experimental techniques in the study of electron-transfer reactions (124). Swaddle and Tregloan recently reviewed electrode reactions of metal complexes in solution at high pressure (125). The main emphasis in this section is on some of the most recent work that we have been involved in, dealing with long-distance electron-transfer processes involving cytochrome c. However, by way of introduction, a short discussion on the effect of pressure on self-exchange (symmetrical) and nonsymmetrical electron-transfer reactions between transition metal complexes that have been reported in the literature, is presented. 

The systems that we investigated in collaboration with others involved intermolecular and intramolecular electron-transfer reactions between ruthenium complexes and cytochrome c. We also studied a series of intermolecular reactions between chelated cobalt complexes and cytochrome c. A variety of high-pressure experimental techniques, including stopped-flow, flash-photolysis, pulse-radiolysis, and voltammetry, were employed in these investigations. As the following presentation shows, a remarkably good agreement was found between the volume data obtained with the aid of these different techniques, which clearly demonstrates the complementarity of these methods for the study of electron-transfer processes. 

In order to obtain further information on the magnitude of the overall reaction volume and the location of the transition state along the reaction coordinate, a series of intermolecular electron-transfer reactions of cytochrome c with pentaammineruthenium complexes were studied, where the sixth ligand on the ruthenium complex was selected in such a way that the overall driving force was low enough so that the reaction kinetics could be studied in both directions (153, 154). The selected substituents were isonicotinamide (isn), 4-ethylpyr-idine (etpy), pyridine (py), and 3,5-lutidine (lut). The overall reaction can be formulated as... 

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