Transforming a blue copper into a red copper protein: Engineering cysteine and homocysteine into the axial position of azurin using site-directed mutagenesis and expressed protein ligation

Interactions of the axial ligand with its blue copper center are known to be important in tuning spectroscopic and redox properties of cupredoxins. While conversion of the blue copper center with a weak axial ligand to a green copper center containing a medium strength axial ligand has been demonstrated in cupredoxins, converting the blue copper center to a red copper center 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 (R _L) of absorption at 447 nm over that at 621 nm. Whereas no axial Cu-S(Cys121) interaction in Met121Cys was detectable by extended X-ray absorption fine structure (EXAFS) spectroscopy at pH 5, similar to what was observed in native 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 R _L 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 a nonproteinogenic amino acid homocysteine that contains an extra methylene group, resulting in a variant whose spectra (R _L= 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 or homocysteine 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 azurin 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.