Reduction of Tryptophan radicals by Tyrosine in proteins

Wild-type metalloproteins were the subject of numerous studies by pulse radiolysis. For instance, one should mention the pioneering work on Cytochrome c (177, 178). Using secondary radicals, it appeared that the reactions with the metal centers almost always involved LRET. Thus several groups tried to modify these proteins in order to bring elements to the determination of electron pathways. 

Another group modified azurin from Pseudomonas aeruginosa, by replacing Metl21 by His. This His residue was used to complex Ru cation (188). The Cu+ to Ru3+ rate constant yielded a P value of l.lA l and the LRET is activationless. 

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. 

The role of the very common a helical structure was investigated as conducting stucture (196, 197, 198). Conclusions are unclear and rather disappointing the a helix structure appears very little conductive. The electron transfer seems to occur through space. 

However maquettes for the design of redox proteins were proposed, based on a three helix bundle with a capping Co(III) (bipyridine)3 electron acceptor at the N-terminus and an electron donor at the C-terminus (199, 200). These proteins were tested for LRET. The a-helical percent was adjusted by addition of urea or trifluoroethanol (201, 202). Intriguingly, studies of one of the proteins (l6-mer-three helix bundle) shows a 2-fold higher LRET rate constant when the percent of helicity is 77% than when it is 0% (denatured in urea). However authors indicate that the kinetics is not a simple first-order one in the presence of urea. They interprete these data as coming from different donor-acceptor distances. The distribution of distances was determined by fluorescence lifetimes fit. Both when helicity is 0% or 77%, distributions peak around 18 A for the Ru(II) (16-mer)3-A (where A=5-((((2-acetyl)amino]ethyl)amino)-naphthalene-l sulfonic acid). Actually the distance appears 0.7A shorter for a-helix which is found consistent with the increased rate constant, by the authors. 

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