Ruthenium-modified proteins

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. 

Using the standard flash photolysis method to produce (NH3)5Ru(II) (His 48)Mb(Fe(III)), both forward and reverse electron transfer was observed in the system 

At 298 K the forward rate constant, ku is 0.019 0.002 s 1 while the reverse constant, kr, is 0.041 0.003 s1. The temperature dependences, in the temperature range 278-318 K, of the forward and reverse rate constants were indeed found to be strong and yielded the activation energy values of 7.4 + 0.5 and 19.5 0.5 kcalmol 1, respectively. 

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Winkler, J. R., and H. B. Gray. Electron transfer in ruthenium-modified proteins. Chem. Rev. 92 (1992), 369-379. 

Figure C3.2.6. Zones associated with the distinctive decay of electronic coupling through a-helical against P-sheet structures in proteins. Points shown refer to specific rates in ruthenium-modified proteins and in the photosynthetic reaction centre. From Gray H B and Winkler J R 1996 Electron transfer in proteins Ann. Rev. Biochem. 65 537.

Binuclear metal complexes often exhibit unusually low ET rates (33, 46, 75). And, at comparable separation distances, the rates of ET in ruthenium-modified proteins are well below those of bridged organic donor-acceptor compounds (90, 118, 126). It is of interest to consider how much of this difference is due to electronic coupling factors. For example, in the complex Os(II)(dioxA)Co(III), A j,(Os to Co) is 7.2 x 10 s (33, 46). Similarly, Ru(BCO)Co has a rate of less than 3.6 x 10 s (4). The Na(Sp)Bi compounds of Closs and Miller having a similar-sized spacer and a smaller driving force have ET rates of approximately 1 x lO s (20,... 

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. 

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 standard ruthenium modification procedure involves the reaction of aquopentaammineruthenium(II) (ajRu " ) with the imidazole of a surface histidine of a protein [5, 13, 14]. The a5Ru(histidine)-modified proteins are stable in both the Ru(II) and the Ru(III) oxidation states and, although ajRu slowly dissociates from surface histidines [15], the ajRu complex stays attached for at least two months under appropriate conditions [16]. 

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... 

Donor-Acceptor Electronic Coupling in Ruthenium-Modified Heme Proteins... 

G. L.C. Moura, I.V. Kurnikov, D.N. Beratan, A. Ponce, A.J. Di Bilio, J.R. Winkler, H.B. Gray, Bond-mediated electron tunneling in ruthenium-modified high-potential iron-sulfur protein, J. Am. Chem. Soc. 

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. 

After a modified protein is prepared, the site of modification must be determined rigorously. It also is necessary to ensure that the structure of the protein has not been perturbed by the modification. Several methods have been used to determine the number and position of metal atoms affixed to the protein surface. The number of metal atoms is commonly determined by atomic absorption analysis (194) or by inductively coupled plasma (ICP) atomic emission analysis (23, 146). Under favorable circumstances, the metal ratios in modified derivatives can be determined by UV/ vis spectroscopy (23, 113). Another method for quantifying ruthenium attached to histidines is to compare the reactions of the native and modified proteins with diethyl pyrocarbonate (146), which is a histidine specific reagent. 

Scheme III. Method for studying Ru" — Fe " electron transfer in ruthenium-modified heme proteins.

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