The Met99Gln mutant of amicyanin from Paracoccus versutus

The axial copper ligand methionine has been replaced by a glutamine in the cupredoxin amicyanin from Paracoccus versutus. Dynamic and structural chara cteristics of the mutant have been studied in detail using UV/Vis, EPR, NMR , cyclic voltammetry, and isomorphous metal replacement. M99Q amicyanin is a blue copper protein with significant spectral and structural similarities to the other cupredoxins umecyanin, stellacyanin, and M121Q azurin. In add ition, the functional properties of M99Q amicyanin, as reflected in the ele ctron self-exchange rate constant and midpoint potential (165 mV), have bee n assessed and compared to values for M121Q azurin. For the latter protein, the published midpoint potential was corrected to the much lower value of 147 mV at pH 7, I = 0.1 M. These values are very similar to the midpoint po tential of stellacyanin, which naturally possesses an axial glutamine ligan d and has the lowest reduction potential for a naturally occurring cupredox in. A remarkable feature of M99Q amicyanin, in the reduced state, is the re latively high pK(a)* value of 7.1 for its His96 ligand.