Ruthenium-modified cytochrome

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]. 

The kinetics of intramolecular electron transfer from Ru(II) to Fe(III) in ruthenium-modified cytochrome c has been studied [77-80]. In these studies electron transfer from electron-excited Ru(II) (bpy)3, which was added to the protein solution, to ruthenium-modified horse heart cytochrome c, (NH3)5Ru(III) (His-33)cyt(Fe(III)), was found to produce (NH3)5Ru(II) (His-33)cyt (Fe(III)) in fivefold excess to (NH3)5Ru(III) (His-33)cyt(Fe(II)). As in refs. 72 and 73, in the presence of EDTA the (NH3)5Ru(II)(His-33)cyt(Fe(III)) decays mainly by intramolecular electron transfer to (NH3)5Ru(III)(His-33)cyt(Fe(II)). The rate constant k — 30 3s 1 at 296 K and does not vary substantially over the temperature range 273-353 K. Above 353 K intramolecular Ru(II) - Fe(III) electron transfer was not observed owing to the displacement of methionine-80 from the iron coordination sphere. The distance of intramolecular electron transfer in this case is also equal to 11.8 A (see Fig. 19). 

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. 

Long range electron-transfer has also been demonstrated within the complex between zinc-substituted cytochrome c peroxidase and cyt c 59). The kinetics of intramolecular electron-transfer from Ru(II) to Fe(III) in ruthenium modified cyt c has also been investigated 58). 

Since their first report of long-ranged electron-transfer reactions in ruthenium-modified cytochrome c [43], Gray and co-workers have studied re-... 

Electron-Transfer Reactions. It is well known that thermal and photochemical electron-transfer reactions exhibit characteristic pressure dependences and associated volumes of activation (see Sections II, III, and VI). It is therefore realistic to expect that photoinduced thermal electron-transfer reactions will also exhibit a characteristic pressure dependence that should reveal mechanistic information on the nature of the reaction. Recent interest in the mechanistic understanding of long-distance electron-transfer reactions prompted an investigation of the effect of pressure on intramolecular electron transfer in ruthenium-modified cytochrome c [151] (a typical example of a closely related intermolecular electron-transfer reaction was... 

The predictive power of pathway analysis is well illustrated with two of the Ru-modified systems of Gray and coworkers [29]. Consider, the His 72 and His 39 ruthenium-modified cytochromes c [28]. The ET rates in these proteins are about the same, despite the fact that the transfer distance is fully 5 A shorter in the His 72 derivative. 

Not all data in the literature are consistent with ET rates scaling with re- Cytochrome C551 has been ruthenated at His-47 the Ru(II)-to-Fe(III) ET rate is 13 for re = 7.9 A. The driving force for ET is the same as for horse heart cytochrome c modified at His-33, and yet ET is slower (13 vs. 30 s ) for an r that is 3.8 A shorter (146). Several other unusually slow rates for short edge-edge separation distances have been observed for ruthenium-modified blue copper and iron-sulfur proteins (see below). 

Conformational changes could control ET reactions in proteins. The rates of such changes often are in the same range as ET rates for example, the T-R transition in hemoglobin occurs at a rate of approximately 2 X 10 s (112). Hoffman and Ratner (66,67) have pointed out that a way to test for conformational control of an ET reaction is to measure the reaction rate at different driving forces. If the rate stays the same, the ET reaction is conformationally controlled. If it does not, it is not confor-mationally controlled. No evidence for conformation control exists for ET in ruthenium-modified proteins on this basis. Data from both ruthenated His-33 in horse heart cytochrome c (126) and ruthenated His-48 in myoglobin (103) show that the rate changes with AG° in a manner consistent with Marcus theory. 

The rate of enzyme turnover is usually determined, not simply by the duration of the substrate-product transformation at the enzyme active center, but by the rate of protein globule relaxation. There is much experimental evidence for this statement. For example, a kinetic and structural study of cytochrome C modified by the ruthenium label specifically at the imidazole moiety of histidine 33, demonstrated that the rate of intramolecular electron transfer, Ru(2) - Haem(3), k 55 s" [56], is on the same time scale as the rate of conformational changes occurring within the cytochrome C molecule [57, 58]. Together with other experimental results (see, for references [38, 39]), this is an obvious indication of the protein conformational changes as the rate-limiting step in protein functioning. 

Forward and reverse rate constants and activation parameters have been determined for the intramolecular electron transfer (13.0 A) in several ruthenium-modified (histidine 39) zinc cytochrome c complexes.These rate constants are 3 times as large as the values for the Ru(His-33) analogs (13.3 A), with slightly greater electronic coupling between the metal centers. The temperature dependence of the rate constant for electron transfer in the Ru(His-33) modified cytochrome c complex has been measured over the 10-200 K range.The rate is independent of temperature (3.6 s" ) between 10 and 100 K, above which two regions of Arrhenius-like behavior (transition at about 150 K) are observed. 

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. 

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... 

Intramolecular electron-transfers through peptides have also been observed by Isied and coworkers using Ru(NH3)5 modified cytochrome c 55). Because of the kinetic inertness of both the ruthenium(II) and ruthenium(III), NMR and other physical techniques can be used to characterize the point of attachment of the ruthenium center. NMR and peptide mapping experiments showed that the ruthenium is bound to the His-33 site of cyt c (Fig. 2). The reduction potentials are +0.26 V for cyt c and +0.07 V for [(NH3)5Ru(His)]2 +. Upon reduction of the Ru(III)-cyt c(III) derivative with 1 equiv. of electrons, any Ru(II)-cyt c(III) produced should undergo... 

Fig. 2. Ruthenium(III) modified cytochrome c(His-33) derivative (from Eisenberg D, Kallai D, Sanson L, Copper A, Margolish E (1971) J. Biol. Chem. 246 1511 and Ref. 55), modified)...

Figure 2.8. The Gibbs energy optimized ET rate vs. edge-to-edge distance relationship for intraprotein electron transfer. The bacteria RC rate constants are shown as circles and excited heme-ruthenium ET in modified myoglobin and cytochrome c are shown as triangles (Moser and Dutton, 1992). Reproduced with permission.

Use of modified gold electrodes is not the only approach to achieve cytochrome c electrochemistry. Indeed, a number of studies have been reported on a variety of electrode surfaces. In 1977, Yeh and Kuwana illustrated (23) well-behaved voltammetric response of cytochrome c at a tin-doped indium oxide electrode the electrode reaction was found to be diffusion-controlled up to a scan rate of 500 mV sec Metal oxide electrodes were further studied (24, 25) independently in Hawkridge and Hill s groups. The electrochemical response of cytochrome c at tin-doped indium oxide and fluoride-doped tin oxide was very sensitive to the pretreatment procedures of the electrode surface. At thin-film ruthenium dioxide electrodes, variation of the faradaic current with pH correlating with the acid-base protonation of the electrode surface was observed. 

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. 

---