Over 30% of proteins in living organisms possess metal ions or organic compounds as a cofactor. Cofactors in proteins participate mainly in biological activities that include oxygen transport, electron transfer, and catalysis. Beside these activities, cofactors play a role in protein folding. Amicyanin has a single copper in their active site as a cofactor and mediates the electron transfer reaction between methylamine dehydrogenase (MADH) and cytochrome c-<sub>551i </sub>for energy production in <i>Paracoccus denitrificans</i>. In this study, the rates of copper incorporation into two different folding states of amicyanin; fully folded apo-amicyanin and partially folded apo-amicyanin, were measured to propose the overall kinetic mechanism for amicyanin biogenesis. When the incorporating copper into partially folded apo-amicyanin, bi-phasic kinetics was observed unlike copper addition to fully folded apo-amicyanin. The rate for the incorporation of copper into fully folded apo-amicyanin was almost 9-fold slower than that for the initial phase of copper addition to partially folded apo-amicyanin. However, it was almost similar to the rate for the second phase of that. Also, when binding affinity of copper on both fully folded apo-amicyanin and partially folded apo-amicyanin was measured by isothermal titration calorimetry (ITC), it was pretty similar each other. These results verify that during amicyanin biogenesis copper rapidly binds to apo-amicyanin before protein folding and then influences the remaining optimization for ligand geometry of copper and the completion of the protein folding reaction.

Thermodynamic and kinetic analysis of amicyanin biogenesis

2019 Fall National Meeting

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