The introduction of a negative charge into the hydrophobic patch of Pseudomonas aeruginosa azurin affects the electron self-exchange rate and the electrochemistry.
By changing Met64 into a glutamate by means of site-directed mutagenesis a negative charge was introduced into the hydrophobic patch of azurin from Pseudomonas aeruginosa. The three-dimensional structure of the protein and the structure of the metal site in particular, appear unaffected by the mutation. The observed change of the midpoint potential of the mutant of 28 mV is ascribed to the deprotonation of Glu64. The electron-self-exchange rate constant equals that of the wild-type protein at pH 4.5 but decreases by almost two orders of magnitude at high pH. Electron transfer is inhibited only when both of the reacting azurin molecules have an ionized glutamate at position 64 in their hydrophobic patch. Electron transfer at a graphite electrode is slowed down by the presence of the negative charge in the hydrophobic patch. The results demonstrate once again that the Cu-ligand His117 in the hydrophobic patch is the likely entry and exit point for electrons. This observation holds both for the (homogeneous) electron-self-exchange reaction in solution as well as for the (heterogenous) reaction at an electrode.
Metallo-protein and Protein engineering Group, Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, The Netherlands.
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Van Pouderoyen
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Mazumdar
N I
NI
Hunt
A O
AO
Hill
G W
GW
Canters
D000595:Amino Acid Sequence; D001400:Azurin / Q000737:chemistry*; D003001:Cloning, Molecular; D004563:Electrochemistry; D004578:Electron Spin Resonance Spectroscopy; D004583:Electrons; D004926:Escherichia coli; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D008715:Methionine; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011199:Potentiometry; D011487:Protein Conformation; D011549:Pseudomonas / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry
D011994:Recombinant Proteins; D001400:Azurin; D008715:Methionine