Pulse radiolysis intramolecular electron transfer

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. 

The intramolecular electron transfer kg, subsequent to the rapid reduction, must occur because the Ru(III)-Fe(II) pairing is the stable one. It is easily monitored using absorbance changes which occur with reduction at the Fe(III) heme center. Both laser-produced Ru(bpy)3 and radicals such as CO (from pulse radiolysis (Prob. 15)) are very effective one-electron reductants for this task (Sec. 3.5).In another approach," the Fe in a heme protein is replaced by Zn. The resultant Zn porphyrin (ZnP) can be electronically excited to a triplet state, ZnP which is relatively long-lived (x = 15 ms) and is a good reducing agent E° = —0.62 V). Its decay via the usual pathways (compare (1.32)) is accelerated by electron transfer to another metal (natural or artificial) site in the protein e. g.. 

Other examples using pulse radiolysis include (a) studies on cytochrome c-di nitrate reductase from Thio-sphaera pantotropha to provide evidence for a fast intramolecular electron transfer from c-heme to rate constant for the reaction of... 

More recently the same reactions have been monitored for Ru(III)-Os(II) analogs. These complexes are generated by the reduction of the Ru(III)—Os(III) dimers by e (aq) or C02 (aq) radicals in pulse radiolysis experiments 429). Because of the much lower inner-sphere reor-ganizational energy terms for the [Ru(NH3)5L]3+/2+ couples compared with Codll/II) analogs, the rates of intramolecular electron transfer in the Ru(III)—Os(II) dimers are much larger than those of Co(III)-Os(II) dimers 429). 

Pulse radiolysis studies on unsymmetrical, chemically linked organic systems have shown the expected fall off in rate constant as AG becomes more favorable.81 In these experiments, advantage is taken of the fact that capture of electrons produced by pulse radiolysis is relatively indiscriminate and in some of the pulse events an electron is captured by the component in the dimeric systems which is the weaker oxidant. Following such an event, the experimental observation made is of the system relaxing by intramolecular electron transfer to the stable redox configuration, as shown for (I)->(2) where A is one of a series of polyaromatic or quinone electron acceptors.81... 

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. 

Kobayashi, K., Koppenh fer, A., Ferguson, S. J., and Tagawa, S., 1997, Pulse radiolysis studies on cytochrome cd nitrite reductase from Thiosphaera pantotropha Evidence for a fast intramolecular electron transfer from c heme to d, heme, RiocAemtstry 36 1361 In 13616. 

Neta and Behar measured the rate constants for intramolecular electron transfer for an extended series of aryl (ArX) and benzyl halide (ArCH2X) radical anions bearing an electron withdrawing ring substituent (NO2, CN, COCH3), generated by pulse radiolysis in aqueous solution (Table 12), conditions under which spectroscopic evidence for formation of the intermediate radical anions was obtained [281-284]. 

Absorption spectra of phenoxyl radicals derived from biologically important molecules were recorded in numerous cases. The tyrosyl radical was studied by many investigators and its spectrum was used to detect tyrosine oxidation in a protein and to follow intramolecular electron transfer from tyrosine to the tryptophan radical in dipeptides and polypeptides . A number of catecholamines, such as adrenaline and dopa, were also studied by kinetic spectrophotometric pulse radiolysis " ". The absorption spectra of most of these substituted o-semiquinone anion were similar to those of the unsubstituted... 

Pulse radiolysis is applied to the measurement of the rate of intramolecular electron transfer between a coordinated ligand-radical and a Co(III) center. The reaction of [Co(NHj)50jCH] + with [OH] and H yields [Co(NH3)5[OjC]-] + which quantitatively yields Co " in <1 fis ... 

The Cu ion reacts with [OH] to form [CuOH] ", which exhibits a pH-depen-dent absorption with = 300 nm. Copper(III) complexes with NHj, ethylene-diamine and various amino acids are characterized by pulse radiolysis . The [Cu(EDTA)] (EDTA = ethylenediaminetetraacetate) and [Cu(NTA)] (NTA = nitrilotriacetate) ions are oxidized to the corresponding Chj(III) species by [OH] Cu(lII) rearranges and then metal-ligand intramolecular electron transfer yields some Cud) species. ... 

Studies of intramolecular ET in oxidases provide interesting examples of how pulse radiolysis is employed to obtain insights into both (1) these enzymes respective mechanisms of action and (2) electron transfer along protein polypeptide matrices that were most probably selected by evolution (9,10, 30-32). Thus, early attempts to study the electron uptake mechanism by the blue oxidase, ceruloplasmin, showed that a diffusion-controlled decay process of the eaq in solutions of this protein is paralleled by the formation of transient optical absorptions due to electron adducts of protein residues, primarily of cystine disulfide bonds (30). The monomolecular decay of the latter absorption was found to have the same rate constant as that at which the type 1 Cu(II) absorption band was reduced. These results were interpreted as being the combined result of the high reactivity of the e q and the relatively inaccessible type 1 Cu(II) site, yielding an indirect, intramolecular electron transfer pathway from surface-exposed residues (30). 

Pulse Radiolysis Measurements of Intramolecular Electron Transfer with Comparisons to Laser Photoexcitation... 

Many clever methods have been developed for generating the thermodynamically unfavored form of the one-electron reduced protein. Details of these methods, which are based on a variety of photochemical and pulse radiolysis techniques, may be found in the original references. The kinetics of intramolecular electron transfer may then be followed by monitoring the rate of approach to the thermodynamically favored state. 

More recently, pulse radiolysis started to play a major role in the characterization of photolytically generated (A —D ) radical pairs in a variety of fullerene containing donor-bridge-acceptor dyads (68,69). While the latter evolve from photoinduced intramolecular electron transfer reactions complementary employment of pulse radiolysis allowed to generate the reduced and oxidized entities in separate experiments and to superimpose the features of the two reactive moieties. In this context, it should be noted that conventional methods, such as cyclic voltammetry, due to their unfavorable time resolution, fail to contribute to the radical pair characterization. 

Pulse radiolysis has been used to study elementary reactions of importance in photosynthesis. Early experiments provided rate constants for electron transfer reactions of carotenoid radical cations and radical anions with chlorophyll pigments.More recent experiments dealt with intramolecular electron transfer in covalently bound carotenoid-porphyrin and carotenoid-porphyrin-quinone compounds. Intramolecular electron transfer reactions within metalloproteins have been studied by various authors much of that work has been reviewed by Buxton, and more recent work has been published. Pulse radiolysis was also used to study charge migration in stacked porphyrins and phthalocyanines. Most of these studies were carried out by pulse radiolysis because this techruque allowed proper initiation of the desired processes and pemtitted determination of very high reaction rate constants. The distinct character of radiolysis to initiate reactions with the medium, in contrast with the case of photolysis, and the recent developments in pulse radiolysis techniques promise continued application of this technique for the study of porphyrins and of more complex chemical systems. 

In peptides and proteins, oxidation of tryptophan is followed by tryptophanyl radical reduction by tyrosine, leading to tyrosinyl radical. This reaction was shown first by Prutz and co-workers (120). Azide radicals are very convenient for this study. This process is easily visualized by pulse radiolysis since both free radicals absorb at different wavelengths (table 3) and the time scale for this reaction goes to microsecond for small peptides to millisecond for proteins. This reaction may occur by intramolecular step and thus it constitutes an excellent model to investigate long range intramolecular electron transfer. These results will be discussed further (see 5.1). 

In the case of PpNiR, heme d can accept an electron without displacement of Tyr25 from the Fe ion and rapid electron transfer is observed. In pulse radiolysis experiments the rate of internal electron transfer was not changed in the presence of nitrite and an intermediate (presumed to be an NO-bound oxidized heme d species) formed within 2 s. Thus the structural differences that result in different rates of primary electron transfer (i.e., the first intramolecular electron transfer following reduction of the oxidized enzyme) appear to be the presence of hydroxide versus Tyr as the axial ligand to heme d. ... 

The kinetics of electron transfer reactions between spinach plastocyanin and [Fe(CN)6] ", [Co(phen)3] , and Fe(II) cytochrome c have been studied as a function of ionic strength. Applications of the equations of Van Leeuwen support the proposal of two sites of electron transfer, with [Co(phen)3] binding near residues 42-45 and the interaction of [Fe(CN)6] at a hydrophobic region near the copper ion. Pulse radiolysis has been employed to measure the rates of electron transfer from Ru(II) to Cu(II) in plastocyanins from Anabaena variabilis and Scenedesmus obliquus which have been modified at His-59 by [Ru(NH3)5] . The small intramolecular rates (<0.082 and <0.26 s , respectively) over a donor-acceptor distance of 12 A indicate that electron transfer from the His-59 site to the Cu center is not a preferred pathway. A more favorable route, via the acidic (residues 42-44) patch ( 14 A to Cu), is supported by the rate of >5 x 10 s for the reduction of PCu(II) by unattached [Ru(NH3)5im] . The intramolecular electron transfer from Fe(II) in horse cytochrome c to Cu(II) in French bean plastocyanin ( 12 A from heme edge to Cys-84 S), in a carbodiimide cross-linked covalent complex, proceeds with a rate of 1.05 x 10 s . The presence of the... 

Tsukahara and Wilkins studied the product of the reaction of CoCNHjjjCmbpy), where mbpy is l-methyl-4,4 -bipyridinium, with COj " at pH 7.2 and 25°C. The COj was formed by pulse radiolysis. The initial product was assigned as the radical complex CoCNHjjjCmbpy ), which then undergoes intramolecular electron transfer (1 = 8.7x10 s" ) and bimolecular electron transfer to Co(NH3)5(mbpy) (1 2 = 5.4x10 s" ). 

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