Intra-electron transfer of amicyanin from newly derived active site to redox potential tuned type 1 copper site

Amicyanin, one of the type I copper proteins which has been used for the study, mediates the electron transfer reaction between methylamine dehydrogenase and cytochrome c-551i in Paracoccus denitrificans for energy production. The 6×Histidine-tag site which has been widely used in purification of a recombinant protein was introduced at the N-terminus of amicyanin to make the complex of 6×His-tagged plus cobalt functioning as a newly derived redox cofactor in amicyanin. In this study, Pro94 of amicyanin was substituted to Ala and Phe to tune up the midpoint potential (E_m) value of amicyanin 100 mV more positive and then intra-electron transfer rates were measured to examine whether the E_m value of the type 1 copper site in amicyanin affects intraprotein electron transfer or not. By the addition of H₂O₂, the Co²⁺-loaded 6×His-tagged site was activated, and then electron was transferred from Cu¹⁺ of type 1 copper site of amicyanin to Co³⁺ plus 6×His-tagged site. Electron transfer rates of cobalt loaded P94A and F amicyanin were much slower than that of native amicyanin. These results suggest that the communication between the newly protein-derived redox cofactor, 6×His-tagged site plus cobalt, and type 1 copper site is truly occurred and that the strength of electron transfer reaction between them is able to be controlled by an E_m value.