The interactions of the axial ligands with copper are known to be important in tuning spectroscopic and redox properties of cupredoxins. While conversion of blue copper sites with a weak axial ligand to green copper sites containing a medium strength axial ligand has been demonstrated in cupredoxins, converting blue copper sites to a red copper site with a strong axial ligand has not been reported. Here we show that replacing Met121 in azurin from Pseudomonas aeruginosa with Cys caused an increased ratio (RL) of absorption at 447 nm over that at 621 nm. While no axial Cu-S(Cys121) interaction in Met121Cys was detectable by the extended x-ray absorption fine structure (EXAFS) at pH 5, similar to what was observed in WT azurin with Met121 as the axial ligand, the Cu-S(Cys121) interaction at 2.74 Å is clearly visible at higher pH. Despite the higher RL and stronger axial Cys121 interaction with Cu(II) ion, the Met121Cys variant remains largely a type 1 copper protein at low pH (with hyperfine coupling constant A|| = 54 × 10−4 cm−1 at pH 4 and 5), or distorted type 1 or green copper protein at high pH (A|| = 87 × 10−4 cm−1 at pH 8 and 9), attributable to the relatively long distance between the axial ligand and copper and the constraint placed by the protein scaffold. To shorten the distance between axial ligand and copper, we replaced Met121 with the nonproteinogenic amino acid homocysteine that contains an extra methylene group, resulting in a variant whose spectra (RL= 1.5, and A|| = 180 × 10−4 cm−1) and Cu-S(Cys) distance (2.22 Å) are very similar to those of the red copper protein nitrosocyanin. Replacing Met121 with Cys resulted in lowering of the reduction potential from 222 mV in the native azurin to 95 ± 3 mV for Met121Cys azurin and 113 ± 6 mV for Met121Hcy at pH 7. The results strongly support the “coupled distortion” model that helps explain axial ligand tuning of spectroscopic properties in cupredoxins, and demonstrate the power of using unnatural amino acids to address critical chemical biological questions.

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The Cu-SCys interaction is known to play a dominant role in defining the type 1 (T1) blue copper center with respect to both its electronic structure and electron transfer function. Despite this importance, its role has yet to be probed by mutagenesis studies without dramatic change of its T1 copper character. We herein report replacement of the conserved Cys112 in azurin with the nonproteinogenic amino acid homocysteine. Based on electronic absorption, electron paramagnetic resonance, and extended x-ray absorption fine structural spectroscopic studies, this variant displays typical type 1 copper site features. Surprisingly, instead of increasing the strength of the Cu-sulfur interaction by the introduction of the extra methylene group, the Cys112Hcy azurin showed a decrease in the covalent interaction between SHcy and Cu(II) when compared with the WT SCys-Cu(II) interaction. This is likely due to geometric adjustment of the center that resulted in the copper ion moving out of the trigonal plane defined by two histidines and one Hcy and closer to Met121. These structural changes resulted in an increase of reduction potential by 35 mV, consistent with lower Cu-S covalency. These results suggest that the Cu-SCys interaction is close to being optimal in native blue copper protein. It also demonstrates the power of using nonproteinogenic amino acids in addressing important issues in bioinorganic chemistry.