Modulating the Copper-Sulfur Interaction in Type 1 Blue Copper Azurin by Replacing Cys112 with Nonproteinogenic Homocysteine.
The Cu-SCysinteraction 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 SHcyand 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-SCysinteraction 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.
University of Illinois-Urbana, Department of Biochemistry, Urbana, IL61801; USA.
Kevin M
KM
Clark
University of Illinois-Urbana. Center for Biophysics and Computational Biology, Urbana, IL 61801, USA.
Yang
Y
Yu
University of Illinois-Urbana, Department of Biochemistry, Urbana, IL61801; USA ; University of Illinois-Urbana. Center for Biophysics and Computational Biology, Urbana, IL 61801, USA ; University of Illinois-Urbana. Department of Chemistry, Urbana, IL 61801, USA ; Howard Hughes Medical Institute, Urbana, IL 61801, USA.
Wilfred A
WA
van der Donk
Oregon Health & Sciences University, Institute of Environmental Health, Beaverton, OR 97006, USA.
Ninian
N
Blackburn
University of Illinois-Urbana, Department of Biochemistry, Urbana, IL61801; USA ; University of Illinois-Urbana. Center for Biophysics and Computational Biology, Urbana, IL 61801, USA ; University of Illinois-Urbana. Department of Chemistry, Urbana, IL 61801, USA.
Yi
Y
Lu