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

Abstract

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.

Effects of mutations on the absorption spectra of copper proteins: a QM/MM study

“…The data fit very well experimental results reported by Clark et al [13], even if care should be taken since in that work azurine protein was used instead, our results being able to correctly reproduce the variation of the absorption spectrum.…”

“…Following the experimental works recently published on azurine by Clark et al [13], we decided to selectively mutate the Met91 residue in spinach plastocyanin to assess for the different spectroscopic properties. The results for some of the most important bands are reported in Table 2.…”

“…Indeed, several such plastocyanins differ from a parent one by a mutation in the coordination arrangement. Finally, artificial mutations can be imposed in order to tune some property of interest [13], or in some cases, equilibrium between green and blue form has been reported [14].…”

“…Recently, the effect of the mutation of the methionine ligand on the absorption spectrum of the blue-copper proteins has been underlined and proved experimentally [13], as well as the artificial mutation of the active site loop sequence of plastocyanin with the nitrosocyanin one giving rise to a red-copper protein [31].…”

Hybrid QM/MM Methods: Treating Electronic Phenomena in Very Large Molecular Systems

“…These two phenomena together induce an important change on the color of the protein and indeed by selectively mutating only one aminoacid, one is able to continuously pass from a blue to a red protein when methionine is substituted with the non proteinogenic aminoacid homocysteine (Hcy) [58]. Note also that these results also nicely reproduce experimental observations [86]. The interaction of organometallic and organic metal with nucleic acid and especially DNA is nowadays a very well recognized phenomenon that is strongly exploited in chemotherapy to induce apoptosis of cancer cells or simply as DNA probe.…”