Effects of dimerization on protein electron transfer

In order to investigate the relationship between the rate of protein-protei n electron transfer and the structure of the association complex, a dimer o f the blue copper protein azurin was constructed and its electron exchange properties were determined. For this purpose, a sire for covalent cross-lin king was engineered by replacing the surface-exposed asparagine 42 with a c ysteine. This mutation enabled the formation of disulfide-linked homodimers of azurin. Based on NMR line-broadening experiments, the electron self-exc hange (e.s.e.) rate constant for this dimer was determined to be 4.2(+/-0.7 ) x 10(5) M-1 s(-1), which is a sevenfold decrease relative to wild-type az urin. This difference is ascribed to a less accessible hydrophobic patch in the dimer. To discriminate between intra molecular electron transfer withi n a dimer and intermolecular electron transfer between two dimers, the e.s. e. rate constant of (Cu-Cu)-N42C dimers was compared with that of (Zn-Cu)- and (Ag - Cu)-N42C dimers. As Zn and Ag are redox inactive, the intramolecu lar electron transfer reaction in these latter dimers can be eliminated. Th e e.s.e. rate constants of the three dimers are the same and an upper limit for the intramolecular electron transfer rate of 10 s(-1) could be determi ned. This rate is compatible with a Cu-Cu distance of 18 Angstrom or more, which is larger than the Cu - Cu distance of 15 Angstrom observed in the wi ldtype crystal structure that shows two monomers that face each other with opposing hydrophobic patches. Modelling of the dimer shows that the Cu-Cu d istance should be in the range of 17 Angstrom < r(Cu-Cu) < 28 Angstrom, whi ch is in agreement with the experimental findings. For efficient electron t ransfer, it appears crucial that the two molecules interact in the proper o rientation. Direct crosslinking may disturb the formation of such an optima l electron transfer complex.