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

Min-Ju Jo,Sooim Shin,Moonsung Choi

doi:10.1007/s13765-018-0344-x

Abstract

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 (Em) value of amicyanin 100 mV more positive and then intra-electron transfer rates were measured to examine whether the Em 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 Em value.

Highlights

Protein engineering techniques have revolutionized many areas of the biological sciences such as chemical processing, bioremediation, and pharmaceuticals
The 69Histidine tag was introduced into the N-terminus amicyanin (Fig. 1A) mediating electron transfer from the methylamine dehydrogenase (MADH) to the cytochrome c-551i in P.denitrificans for bioenergetic processes [2,3,4]
The electron transfer reaction from cobalt bound to 69His tag site to copper in the active site of amicyanin was measured in 0.05 M Tris– HCl buffer at pH7.5. 100 lM of CoCl2 (Sigma-Aldrich) was used to treat 40 lM of 69His-tagged and non-tagged amicyanins for 30 min, and absorbance changes were monitored at 330 nm

Summary

Introduction

Protein engineering techniques have revolutionized many areas of the biological sciences such as chemical processing, bioremediation, and pharmaceuticals. 3 Department of Optometry, College of Energy and Biotechnology, Seoul National University of Science and Technology, Seoul 01811, Republic of Korea communication between the newly protein-derived redox cofactor, 69His-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 Em value. Adding cobalt to a 69-Histidine tag, which is generally used for recombinant protein purification, plus H2O2 mediates intra-electron transfer in amicyanin [1].

Results

Conclusion