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The axial copper ligand methionine has been replaced by a glutamine in the cupredoxin amicyanin from Paracoccus versutus.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 characteristics 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 addition, the functional properties of M99Q amicyanin, as reflected in the electron self-exchange rate constant and midpoint potential (165 mV), have been 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 potential of stellacyanin, which naturally possesses an axial glutamine ligand and has the lowest reduction potential for a naturally occurring cupredoxin. A remarkable feature of M99Q amicyanin, in the reduced state, is the relatively high pK(a) value of 7.1 for its His96 ligand.
Gorlaeus Laboratories, Leiden Institute of Chemistry, Leiden University, The Netherlands.
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Dennison
D019943:Amino Acid Substitution / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D003035:Cobalt / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004579:Electron Transport / Q000235:genetics; D005973:Glutamine / Q000235:genetics*; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008715:Methionine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D010230:Paracoccus / Q000737:chemistry* / Q000235:genetics*; D011725:Pyridines / Q000737:chemistry; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D019163:Reducing Agents / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013439:Sulfhydryl Reagents / Q000737:chemistry
D001426:Bacterial Proteins; D004220:Disulfides; D011725:Pyridines; D011994:Recombinant Proteins; D019163:Reducing Agents; D013439:Sulfhydryl Reagents; C017079:mauC protein, Methylobacterium extorquens; D005973:Glutamine; C007542:4,4'-dipyridyl disulfide; D003035:Cobalt; D006639:Histidine; D008715:Methionine