Electronic coupling azurin

The type-1 blue copper proteins act as electron carriers azurin, plastocyanin, stellacyanin, umecyanin e.g. They are characterized by a rather strong LMCT (ligand to metal charge transfer) band near 600 nm and by small hyperline coupling constants A in EPR. Copper is bound to two imidazole groups of histidine and to two... 

The possibility of introducing single-site mutations in azurins enabled a detailed analysis of structure-reactivity relationships where, for example, the impact of specific amino acid substitutions on the rate of intramolecular FT could be investigated. In order to understand better the role of the polypeptide matrix separating electron donor and acceptor on FT reactivity, the structure-dependent theoretical model developed by Beratan et al. (6, 7) was employed to identify relevant ET pathways (cf. Section I.B). In this model, the total electronic coupling of a pathway is calculated as a repeated product of the couplings of the individual links. The optimal pathway connecting the two redox sites, is thus identified (cf. Eq. 5). 

The entropy of activation, which includes a contribution from the separation distance dependence of the electronic coupling is given by Eq.7. It is seen that the increase in rate in V31W azurin follows from a more favorable entropy of activation (Table I), which is larger by 16.8JK mol compared with WT azurin. Since AS° can safely be assumed to be the same for intramolecular ET in... 

Figure 6. Electron-transfer pathway in the azurin dimer mutant (43). The path connects Sy of Cys42 with Nj of His46, which is one of the copper ligands, and consists of 17 covalent bonds resulting in a very effective electronic coupling of the two redox centers. Calculations were based on the Beratan and Onuchic model (6, 7). Coordinates were taken from the PDB, code IJVO.

Figure 6. Sets of pathway tubes for the ET coupling from Cu to Ru(bpy)2(im) in HIS 126-modified azurin. The shaded curves (dashed lines are H-bonds) indicate the cores of the tubes that together are responsible for effectively all the electronic coupling of the protein matrix. Nos. 122 and 124 are like 126, but with a subset of the tubes shown here. The copper ligands are 46, 117, and 112, and the coupling is dominated by the 112 fi-strand.

Pseudomonas aeruginosa (P.a.) azurin has been ruthenated at His-83 (r 11.8 A) (54, 93, 113) the donor-acceptor separation is pictured in Fig. 19. Production of a5Ru(His-83) -Az(Cu ) was achieved by flash photolysis in the presence of [Ru(bpy)3]. The reduction of the protein was monitored at 625 nm, and the intramolecular Ru(II)-to-Cu(II) ET rate of 1.9 s was found to be independent of temperature. The Cu reorganization enthalpy was estimated to be <7 kcal mol (93, 113), a value confirming that blue copper is structured for efficient ET. Table XI compares ET rates for the blue copper proteins with those for heme proteins the blue copper rates are low in comparison with the heme protein rates over similar distances and driving forces. This effect could be a result of poor electronic coupling of asRu with the copper center, possibly owing to unfavorable ET pathways. 

As an example on the relationship between proton relaxivity, electron relaxation and coordination environment, we report the case of azurin and its mutants. The relaxivity of wild type azurin is very low (Fig. 6) due to a solvent-protected copper site, the closest water being found at a distance of more than 5 A from the copper ion. The fit, performed with the Florence NMRD program, able to take into account the presence of hyperfine coupling with the metal nucleus (Ay = 62 x 0 cm , see Section II.B) indicates Tie values of 8 X 10 s. Although the metal site in azurin is relatively inaccessible, several mutations of the copper ligands open it up to the solvent. The H NMRD profiles indicate the presence of water coordination for the... 

The electron transfer mechanism of azurin, a well known example for this type of proteins, has been systematically studied using the chemical relaxation method and a well defined inorganic outer sphere redox couple. In parallel, the investigations of the reaction with its presumed physiological partner, cytochrome c, were pursued (7). The specificity of the interaction between azurin and cytochrome c P-551 is expressed in higher specific rates and in the control of the electron transfer equilibrium by conformational transitions of both proteins. 

An effective approach to resolving the electron pathway to and from the redox center of azurin is the systematic investigation of its equilibria and kinetics of interactions with inorganic redox couples. Hexacyano-ferrate (II/III) is a well defined redox couple, known to react via an... 

As the axial ligand is weakly bound in BCP (Randall et al., 2000), the spin density delocalized on it is small. Indeed, in azurin the resonances of the axial methionine protons do not experience a significant hyperfine shift contribution. Electron delocalization onto a Hy of the axial Met has been detected in plastocyanin (signal F in Fig. 3B), suggesting some covalency for the Cu-S(Met) bond. The absence of spin density on the axial Gin ligand in stellacyanin has been attributed to the fact that the y-CH2 Gin geminal couple is four bonds away from the metal ion, whereas the equivalent protons in a bound Met residue (such as in plastocyanin) are only three bonds away (Bertini et al., 2000). 

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