Ruthenium complexes cytochrome

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... 

A similar study was performed on ruthenium-modified myoglobins, in which AG variations were obtained by changing the nature of the ruthenium complex covalently bound to the protein, and by substituting a porphyrin to the heme [137]. It is gratifying to observe that, in spite of the rather heterogeneous character of this series, the study leads to an estimation of 1.9 to 2.4 eV for A which is consistent with the value 2.3 eV derived in section 3.2.1 from temperature dependent experiments. Satisfactory agreement between the results given by the two methods is also observed in the case of ruthenium-modified cytochrome c [138]. 

A variety of physical methods has been used to ascertain whether or not surface ruthenation alters the structure of a protein. UV-vis, CD, EPR, and resonance Raman spectroscopies have demonstrated that myoglobin [14, 18], cytochrome c [5, 16, 19, 21], and azurin [13] are not perturbed structurally by the attachment of a ruthenium complex to a surface histidine. The reduction potential of the metal redox center of a protein and its temperature dependence are indicators of protein structure as well. Cyclic voltammetry [5, 13], differential pulse polarography [14,21], and spectroelectrochemistry [12,14,22] are commonly used for the determination of the ruthenium and protein redox center potentials in modified proteins. 

Intramolecular electron transfer from Ru(II) to Fe(III) in (NH3)3Ru(II) (His-33)cyt(Fe(III)) induced by pulse-radiolysis reduction of Ru(III) in the (NH3)5Ru(III) (His-33)cyt(Fe(III)) complex were investigated [84]. The results obtained differ from those of refs. 77-80 where flash photolysis was used to study the similar electron transfer reaction. It was found [84] that, over the temperature range 276-317 K the rate of electron transfer from Ru(II) to Fe(III) is weakly temperature dependent with EA 3.3 kcal mol 1. At 298 K the value of kt = 53 2 s"1. The small differences in the temperature dependence of the electron tunneling rate in ruthenium-modified cytochrome c reported in refs. 77-80 and 84 was explained [84] by the different experimental conditions used in these two studies. 

Iron-containing cytochrome P-450 constitutes the most famous example of a selective C-H bond oxidizer. Although the exact nature of the mechanism remains controversial, the reaction most likely proceeds through radical intermediates [2]. The hydroxylation of activated C-H bonds has also been carried out in the presence of synthetic porphyrin complexes. In these biomimetic processes, ruthenium plays a relatively minor role when compared with iron. Zhang et al. [50], however, recently reported the enantioselective hydroxylation of benzylic C-H bonds using ruthenium complexes supported by a D4-sym-metric porphyrin bearing a crafted chiral cavity. Thus, complex 23 reacts in a stoichiometric manner with ethylbenzene to give phenethyl alcohol with a... 

Engstrom G, Rajagukguk R, Saunders A, et al. Design of a ruthenium-labeled cytochrome c derivative to study electron transfer with the cytochrome be, Complex. Biochemistry 2003 42 2816-24. 

Selective oxidative demethylation of tertiary methyl amines is one of the specific and important functions of cytochrome P-450. Novel cytochrome P-450-type oxidation behavior with tertiary amines has been found in the catalytic systems of low-valent ruthenium complexes with peroxides. These systems exhibit specific reactivity toward oxidations of nitrogen compounds such as amines and amides, differing from that with RUO4. It was discovered in 1988 that low-valent ruthenium complex-catalyzed oxidation of tertiary methylamines 53 with f-BuOOH gives the corresponding a-(f-butyldioxy)alkylamines 54 efficiently (Eq. 3.70) [130]. The hemiaminal type 54 product has a similar structure to a-hydroxymethylamine intermediate derived from the oxidation with cytochrome P-450. 

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. 

Materials. The preparation of the H39C C102T variant of yeast cytochrome c has been described by Hilgen and Pielak (19). Reaction of this variant with Ru(bpy)2(BrCH2bpyCH3) and subsequent purification and characterization of the labeled protein have been described by Geren et al. (20, 21). The other derivatives were prepared by analogous methods. Preparation of the ruthenium complexes has been described by Scott et al. (9) and Ernst and Kaim (22). 

Table I. Rate Constants for Intramolecular Electron Transfer in H39C C102T Variant of Yeast Cytochrome c Covalently Bound to Ruthenium Complexes at Cys39.

The observed rate constants for intramolecular electron transfer between the heme center of cytochrome c and covalently bonded ruthenium complexes appear to be true measures of rates of intramolecular electron transfer despite the independence of rate from the free energy of reaction. The calculated value... 

Table III. Summaiy of Rate and Activation Parameters for the Electron Transfer Reaction Between Cytochrome c and Several Pentammine-Ruthenium Complexes Ru asL + cyt Ru asL + cyt...

Figure 11 Molecular model of the complex between Ru-65-cyt i>5 and Cc. The geometry of the complex is the same as that of the complex involving native cytochrome proposed by Salemme. The heme groups (red), and the ruthenium complex (green) are highlighted. The atoms forming an electron-transfer pathway between the ruthenium complex and the heme group of Ru-65-cyt hs are colored yeUow. The lysine and arginine residues are blue, while aspartate and glutamate residues are red ...

Intramolecular oxidation and reduction in cytochrome c complexes covalently modified was studied by several groups (for review see 190). Histidines (191, 192, 193) and cysteines (194) were used to attach covalently Ruthenium complexes to Fe- or Zn-substituted cytochrome c. Most of the experiments were done using laser lash photolysis. In each series of experiments, the distance was considered as constant and determined by molecular modelling. The free energies span between 0.5 to 1.4V. The L T rate constants do vary with the driving force as expected. However the reactions proceed with rate constants lower than those expected on the basis of results obtained on peptides. Results were all analyzed using Marcus theory. X and Hab were considered as adjustable parameters. Each series of experimental data was fitted separately (3 to 6 points). In all these papers, X values go from 1.15 to 1.22 eV and Hab vary from 0.1 to 0.24 cm l. Activation volumes were also measured (195). It seems that the transition state is more compact than the reactant state in both intra- and inter-molecular steps. 

This general approach has, however, serious limitations. The position of the site for attack (and therefore the electron transfer distance involved) is very conjectural. In addition, the vexing possibility, which we have encountered several times, of a dead-end mechanism (Sec. 1.6.4) is always present. One way to circumvent this difficulty, is to bind a metal complex to the protein at a specific site, with a known (usually crystallographic) relationship to the metal site. The strategy then is to create a metastable state, which can only be alleviated by a discernable electron transfer between the labelled and natural site. It is important to establish that the modification does not radically alter the structure of the protein. A favorite technique is to attach (NH3)5Ru to a histidine imidazole near the surface of a protein. Exposure of this modified protein to a deficiency of a powerful reducing agent, will give a eon-current (partial) reduction of the ruthenium(III) and the site metal ion e.g. iron(III) heme in cytochrome c... 

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