Serine Mutation on Amicyanin Reveals Functional and Structural Roles of Asn47 and Asn54 in the Cu-Binding Ligand Containing Loop

Amicyanin is a type 1 Cu protein that mediates electron transfer between methylamine dehydrogenase and cytochrome c-551i in Paracoccus denitrificans. In this study, a Ser mutation was introduced at either Asn47 or Asn54 located in the Cu-binding ligand loop containing His53 to determine their role in amicyanin functionality. Their spectral and redox properties, and protein stability according to temperature variance and oxidative stress were investigated. N47S amicyanin indicated similar redox potential and stability to native amicyanin. The reaction kinetic of N47S amicyanin toward methylamine dehydrogenase exhibited a similar electron transfer rate, but immensely improved binding affinity compared to native amicyanin. For N54S amicyanin, it attained a more positive redox potential and greatly reduced stability. N54S amicyanin also altered the reaction kinetics with increased electron transfer and decreased binding affinity. Combined with these results, computational simulations of the N47S and N54S mutations suggest that the Ser substitution at Asn54 alters the geometry of the Cu active site by changing the surrounding H-bond pattern. On the other hand, although N47S did not affect the active site, it can be deduced that the position of Asn47 in the loop is significantly altered to influence the interaction of amicyanin with MADH. Hence, we conclude that Asn47 takes charge of the amicyanin affinity for MADH while Asn54 regulates the electron transfer by altering the redox midpoint potential of the active site.