Modulating the copper–sulfur interaction in type 1 blue copper azurin by replacing Cys112 with nonproteinogenic homocysteine

Abstract

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.

Design and fine-tuning redox potentials of metalloproteins involved in electron transfer in bioenergetics

“…Replacement of the Cys ligand in the Zn-binding site of the same protein to a His resulted in a green T1Cu site [64,65,67]. Both green and red T1Cu sites were engineered in the blue T1 site of azurin (Az) through incremental increase of strength to the axial Met by mutating it to a Cys or unnatural Hcy (homocysteine), respectively [68,69] (Figure 4b). …”

Effect of circular permutation on the structure and function of type 1 blue copper center in azurin

“…This minimized energy status is known as the entatic or "rack-induced" state. 1,19,20 While the roles of primary coordination sphere (i.e., ligands that coordinate to the metal center directly) and secondary coordination sphere residues (i.e., residues that interact with the primary coordinating ligands) in tuning reduction potentials and reorganization energies of the T1 copper proteins have been extensively studied by site-directed mutagenesis and unnatural amino acids incorporation, [21][22][23][24][25][26][27][28][29][30] the impact of the changing the overall protein fold on the "rack-induced" state has not been explored. To address this issue, we report herein construction and characterization of circularly permutated variants of azurin, a T1Cu protein, and investigation of the effects of protein scaffold changes on both structural and functional properties of the T1Cu center.…”

A Purple Cupredoxin from Nitrosopumilus maritimus Containing a Mononuclear Type 1 Copper Center with an Open Binding Site

“…[11][12][13][14][15] Mononuclear cupredoxins have been studied for decades and almost all of them natively contain coordinately saturated Cu site and perform ET function. [1][2][3][4][5][6][7][8][9][10]13,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] The only exception so far is nitrosocyanin, which is in a cupredoxin scaffold but contains an additional water ligand. 31,32 Spectroscopic and x-ray crystallographic studies suggested that nitrosocyanin contains a type 2 copper (T2Cu) center, similar to many copper enzymes.…”