Through the use of site-directed mutagenesis, it has been possible to replace th

Question

Through the use of site-directed mutagenesis, it has been possible to replace the cysteine bound to copper in azurin with aspartic acid. What differences in the spectroscopic properties would you predict for the copper(II) complex of the mutant versus the wild-type protein? Through the use of site-directed mutagenesis, it has been possible to replace the cysteine bound to copper in azurin with aspartic acid. What differences in the spectroscopic properties would you predict for the copper(II) complex of the mutant versus the wild-type protein?

Explanation / Answer

The intense charge transfer band in the 600 nm region characteristic of a blue copper site is conspiciously absent in the absorption spectrum of Cu(II) complex of mutant azurin. Furthermore, an absorption band not seen in wild type azurin is observed as a shoulder to the red of the near-UV protein band. This band is attributable to an imidazole-to-cu(II) charge transfer transition in a tetragonal coordination site, and is blue shifted considerably from a related ligand-to-metal charge transfer absorption at 481 nm in cu(II) wild type azurin. The inferred tetragonal structure of cu(II) in mutant in the EPR spectrum is attributable to a normal type 2 copper centre. The EPR spectrum of cu(II) complexed mutant protein exhibits superhyperfine structure in the lowest hyperfine line indicating the presence of two equivalent equatorial nitrogen ligands.

To conclude, the absolute requirement for a blue site is Cys. When this is changed to aspartic acid, the protein still binds Cu(II) strongly, but the visible and EPR spectra are characteristic of tetragonal (type 2 or normal) geometry rather than of a type 1 site

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