An intramolecular electron-transfer process has previously been shown to take place between the Cys3 - Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single... Show moreAn intramolecular electron-transfer process has previously been shown to take place between the Cys3 - Cys26 radical-ion (RSSR-) produced pulse radiolytically and the Cu(II) ion in the blue single-copper protein, azurin [Farver, O. & Pecht, I. (1989) Proc. Natl Acad Sci. USA 86, 6868 - 6972]. To further investigate the nature of this long-range electron transfer (LRET) proceeding within the protein matrix, we have now investigated it in two azurins where amino acids have been substituted by single-site mutation of the wild-type Pseudomonas aeruginosa azurin. In one mutated protein, a methionine residue (Met44) that is proximal to the copper coordination sphere has been replaced by a positively charged lysyl residue ([M44K]azurin), while in the second mutant, another residue neighbouring the Cu-coordination site (His35) has been replaced by a glutamine ([H35Q]azurin). Though both these substitutions are not in the microenvironment separating the electron donor and acceptor, they were expected to affect the LRET rate because of their effect on the redox potential of the copper site and thus on the driving force of the reaction, as well as on the reorganization energies of the copper site.The rate of intramolecular electron transfer from RSSR - to Cu(II) in the wild-type P. aeruginosa azurin (DELTAG-degrees = -68.9 kJ/mol) has previously been determined to be 44 +/- 7 s-1 at 298 K, pH 7.0. The [M44K]azurin mutant (DELTAG-degrees = - 75.3 kJ/mol) was now found to react considerably faster (k = 134 +/- 12 s-1 at 298 K, pH 7.0) while the [H35Q]azurin mutant (DELTAG-degrees = - 65.4 kJ/mol) exhibits, within experimental error, the same specific rate (k = 52 +/- 11 s-1 298 K, pH 7.0) as that of the wild-type azurin. From the temperature dependence of these LRET rates the following activation parameters were calculated: DELTAH double dagger = 3 7.9 +/- 1.3 kJ/mol and 47.2 +/- 0.7 kJ/mol and DELTAS double dagger = - 86.5 +/- 5.8 J/mol . K and - 46.4 +/- 4.4 J/mol . K for [H35Q]azurin and [M44K]azurin, respectively. Using the Marcus relation for intramolecular electron transfer and the above parameters we have determined the reorganization energy, lambda and electronic coupling factor, beta. The calculated values fit very well with a through-bond LRET mechanism. Show less
Kamp, M. van de; Canters, G.W.; Wijmenga, S.S.; Lommen, A.; Hilbers, C.W.; Nar, H; ... ; Huber, R. 1992
Complete sequential H-1 and N-15 resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) 14 000, 128 residues) from Pseudomonas aeruginosa have been obtained at pH... Show moreComplete sequential H-1 and N-15 resonance assignments for the reduced Cu(I) form of the blue copper protein azurin (M(r) 14 000, 128 residues) from Pseudomonas aeruginosa have been obtained at pH 5.5 and 40-degrees-C by using homo- and heteronuclear two-dimensional (2D) and three-dimensional (3D) nuclear magnetic resonance spectroscopic experiments. Combined analysis of a 3D homonuclear H-1 Hartmann-Hahn nuclear Overhauser (3D H-1 HOHAHA-NOESY) spectrum and a 3D heteronuclear H-1 nuclear Overhauser H-1{N-15} single-quantum coherence (3D H-1{N-15} NOESY-HSQC) spectrum proved especially useful. The latter spectrum was recorded without irradiation of the water signal and provided for differential main chain amide (NH) exchange rates. NMR data were used to determine the secondary structure of azurin in solution. Comparison with the secondary structure of azurin obtained from X-ray analysis shows a virtually complete resemblance; the two beta-sheets and a 3(10)-alpha-3(10) helix are preserved at 40-degrees-C, and most loops contain well-defined turns. Special findings are the unexpectedly slow exchange of the Asn-47 and Phe-114 NH's and the observation of His-46 and His-117 (NH)-H-epsilon2 resonances. The implications of these observations for the assignment of azurin resonance Raman spectra, the rigidity of the blue copper site, and the electron transfer mechanism of azurin are discussed. Show less
Nar, H.; Messerschmidt, A.; Huber, R.; Kamp, M. van de; Canters, G.W. 1991
