Enhanced rate of intramolecular electron transfer in an engineered purple CUA azurin

The recent expression of an azurin mutant where the blue type 1 copper site is replaced by the purple Cu-A site of Paracoccus denitrificans cytochrome c oxidase has yielded an optimal system for examining the unique electron mediation properties of the binuclear Cu-A center, because both type 1 and Cu-A centers are placed in the same location in the protein while all other structural elements remain the same, Long-range electron transfer is induced between the disulfide radical anion, produced pulse radiolytically, and the oxidized binuclear Cu-A center in the purple azurin mutant. The rate constant of this intramolecular process, k(ET) = 650 +/- 60 s(-1) at 298 K and pH 5.1, is almost 3-fold faster than for the same process in the wild-type single blue copper azurin from Pseudomonas aeruginosa (250 +/- 20 s(-1)), in spite of a smaller driving force (0.69 eV for purple Cu-A azurin vs, 0.76 eV for blue copper azurin), The reorganization energy of the Cu-A center is calculated to be 0.4 eV, which is only 50% of that found for the wild-type azurin, These results represent a direct comparison of electron transfer properties of the blue and purple Cu-A sites in the same protein framework and provide support for the notion that the binuclear purple Cu-A center is a more efficient electron transfer agent than the blue single copper center because reactivity of the former involves a lower reorganization energy.