Redox proteins, pulse radiolysis

Pecht, I., Goldberg, M. Electron transfer pathways to and within redox proteins Pulse radiolysis studies. In Fast Processes in Radiation Chemistry and Biology (Adams, G. E., Fielden, E. M., Michael, B. D., eds.), Wiley, Chichester-New York-Toronto-Sydney, 1975, pp. 277-284... 

Flash photolysis and pulse radiolysis techniques have been developed to study Fe Ru ET in Ru-modified proteins [21,26,27]. A method that allows study of electron transfer from a surfaee ajRu(IIIXhistidine) to a protein redox center is outlined in the Scheme [21]. The ET reaction is initiated by photogenerated... 

Metalloproteins, where the active site includes one or more metals, represent a very different class of proteins than those discussed above. The particular kinds of metalloproteins discussed here are those where the metal is redox active and represents a functional and not structural component of the system. Many mechanistic studies of metalloproteins have been carried out using radiation chemistry in the past 50 years. Two different ways of using radiation chemistry to query mechanisms will be illustrated here. The first, as described in the earliest of these studies using blue copper proteins such as azurin, involves using pulse radiolysis to change an oxidation state and thus... 

Elaboration of LRET mechanism by resolving the parameters that determine specific rates of LRET has stimulated pulse radiolysis studies in proteins. Examples include generation of metastable electron donor and acceptor complexes in (1) native and mutant proteins, (2) proteins with the directed single-site specific mutations, (3) native and mutant multisite redox proteins, (4) proteins with the site specific modification with transition metal complexes covalently attached to a specific surface amino acid residues. 

Haem peroxidases are globular proteins with an iron-porphyrin complex as a prosthetic group. These enzymes are widespread among prokaryotes and eukaryotes. They catalyze the oxidation of substrates by organic peroxides or hydrogen peroxide. During the past decades, considerable scientific effort has been put into elucidation ofthe mechanisms of reactions catalyzed by these enzymes. Pulse radiolysis technique has made an important contribution by providing information on the redox states of the enzymes and their interconversion, as well as on the properties ofthe free radical intermediates involved [23]. 

El Hanine, Lmoumene C., Conte D., Jacquot J.-R, Houee-Levin C., Redox properties of protein disulfide bond in oxidized thioredoxin and lysozyme. A pulse radiolysis study. Biochemistry, 2000,39,9295-9301. 

This chapter reviews results and current insights emerging primarily from pulse radiolysis (PR) studies of intramolecular ET in multisite proteins, mainly iron- and copper-containing redox enzymes, with emphasis on interactions between the different redox centers. 

For these reactions, AG° 0. The experimental measurement of cross-reaction rates is generally more straightforward than the measurement of self-exchange rates. Either the reactants are simply mixed together, or a thermodynamically unstable system is generated rapidly (via pulse radiolysis, flash photolysis, or temperature-jump relaxation) to initiate the redox reaction. Absorption spectroscopy has almost always been used to monitor the progress of protein cross reactions. The primary goal of theory, as will become evident, is to provide a relationship between AG° and AG" " for cross reactions. 

The potential of pulse radiolysis for studying biological redox processes, particularly of macromolecules, was recognized rather early. It was initially employed for investigating radiation-induced damage and, later on, as an effective tool for resolving electron transfer processes to and within proteins. Cytochrome c, a well-characterized electron-mediating protein, was the first to be... 

Pulse radiolysis has been employed successfully to resolve mechanisms of action of redox proteins and of electron transfer within their polypeptide matrix. The limitations on the use of this method, set by the requirement for expensive electron accelerators, are more than compensated for by experimental advantages, as illustrated by the results described in this chapter. Future applications to the study of engineered proteins and other model systems would certainly extend our understanding of both of these aspects of redox processes in biological macromolecules. 

An alternative approach to the generation of suitable protein-bound redox was also investigated. Nitration of surface Tyr residues in CCP was carried out to generate protein-bound reducing N02 -Tyr radicals in situ (28), and our preliminary results are provided in the section Pulse Radiolysis Studies of CCP/ Finally, the use of flash photolysis and pulse radiolysis techniques in the study of Fe =0 heme systems is compared. 

We prepared three bifunctional redox protein maquettes based on 12 16-, and 20-mer three-helix bundles. In each case, the helix was capped with a Co(III) tris-bipyridyl electron acceptor and also functionalized with a C-terminal viologen (l-ethyl-V-ethyl-4,4 -bipyridinium) donor. Electron transfer (ET) was initiated by pulse radiolysis and flash photolysis and followed spectrometrically to determine average, concentration-independent, first-order rates for the 16-mer and 20-mer maquettes. For the 16-mer bundle, the a-helical content was adjusted by the addition of urea or trifluoroethanol to solutions containing the metal-loprotein. This conformational flexibility under different solvent conditions was exploited to probe the effects of helical secondary structure on ET rates. In addition to describing experimental results from these helical systems, this chapter discusses several additional metalloprotein models from the recent literature. 

As with donor-acceptor model complexes, ET reactions in proteins have been induced by photoexcitation and pulse radiolysis. In pulse radiolysis experiments, it has been found that the ratio of reduction of the surface metal center to the internal protein redox center can be manipulated, depending on the choice of the mediator. With a5Ru "(His-33)Fe" cytochrome c, reduction of the Ru(III) site was 35% efficient with isopropanol as mediator and 95% efficient with pentaerythritol as mediator (74). 

The reduction of ferricytochrome c by hydrated electrons and by several free radicals has been studied by pulse radiolysis. The reduction of oxidized cytochrome c by [Fe(edta)] - follows first-order kinetics for both protein and reductant, with a rate constant of 2.57 x 10 1 mol" s" at pH 7 and activation enthalpy and entropy of 6.0 kcal mol" and —18 cal K" mol", respectively. These values are comparable to those for outer-sphere cytochrome c reductions and redox reactions involving simple iron complexes, and are compatible with outer-sphere attack of [Fe(edta)] " at the exposed haem edge, although the possibility of adjacent attack through the haem pocket is not ruled out. The rate data at pH 9 are consistent with [Fe(edta)] " reduction of two slowly interconverting forms of the protein, native kt = 2.05 X10 1 mol" S" ) and high-pH kt = 2.67 x 10 1 mol" s" ) isomers. A possible route for the transfer of the electron from Cr + to ferricytochrome c has been suggested as a result of the chemical analysis of the chromium(m) product. The reduction by Cr + of the native protein and of ferricytochrome c carboxy-methylated at the haem-linked methionine (residue 80) has been studied kinetically. At pH 6.5 the former process is simple and corresponds to a second-order rate constant of 1.21 x 10 1 mol" s". The latter, however, is complex - two chromium-... 

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