Effects of Dimerization on Protein Electron Transfer

In order to investigate the relationship between the rate of protein–protein electron transfer and the structure of the association complex, a dimer of the blue copper protein azurin was constructed and its electron exchange properties were determined. For this purpose, a site for covalent cross‐linking was engineered by replacing the surface‐exposed asparagine 42 with a cysteine. This mutation enabled the formation of disulfide‐linked homodimers of azurin. Based on NMR line‐broadening experiments, the electron self‐exchange (e.s.e.) rate constant for this dimer was determined to be 4.2(±0.7)×10 5   M −1  s −1 , which is a seven‐fold decrease relative to wild‐type azurin. This difference is ascribed to a less accessible hydrophobic patch in the dimer. To discriminate between intramolecular electron transfer within 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 intramolecular electron transfer reaction in these latter dimers can be eliminated. The 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 determined. This rate is compatible with a Cu–Cu distance of 18 Å or more, which is larger than the Cu–Cu distance of 15 Å observed in the wild‐type crystal structure that shows two monomers that face each other with opposing hydrophobic patches. Modelling of the dimer shows that the Cu–Cu distance should be in the range of 17 Å< r Cu–Cu <28 Å, which is in agreement with the experimental findings. For efficient electron transfer, it appears crucial that the two molecules interact in the proper orientation. Direct cross‐linking may disturb the formation of such an optimal electron transfer complex.