Abstract
Glutaredoxins (GRXs) are a class of enzymes used in the reduction of protein thiols and the removal of reactive oxygen species. The CPYC active site of GRX is a plausible metal binding site, but was previously theorized not to bind metals due to its cis-proline configuration. We have shown that not only do several transition metals bind to the CPYC active site of the Brucella melitensis GRX but also report a model of a dimeric GRX in the presence of silver. This metal complex has also been characterized using enzymology, mass spectrometry, size exclusion chromatography, and molecular modeling. Metalation of GRX unwinds the end of the helix displaying the CPYC active site to accommodate dimerization in a way that is similar to iron sulfur cluster binding in related homologs and may imply that metal binding is a more common occurrence in this class of oxidoreductases than previously appreciated.
Highlights
Glutaredoxins (GRXs) are proteins that utilize dithiol reduction for maintenance of the intracellular redox state [1]
GRX’s redox centers consist of either a dithiol active site (CPYC) or a monothiol active site (CGFS) with the former typically associated with oxidoreductase reactions and the latter principally associated with iron sulfur cluster formation/maturation [1,2,3,4]
This begs the question why do metal ions prefer the configuration of the CXXC motif in metallochaperones, as opposed to the CPYC site in GRX and other thioredoxin fold proteins? One hypothesis for this specificity that had been proposed is that the proline in the GRX active site prevents metal binding and/or the dimerization required for metal transfer [14]
Summary
Glutaredoxins (GRXs) are proteins that utilize dithiol reduction for maintenance of the intracellular redox state [1]. The interaction of ATP7a and ATP7b with GRX remains dependent on the concentration of copper [12] and knockdown of GRX1 in drosophila disrupts copper homeostasis [13] The thiols in these proteins are required for both redox maintenance and metal binding. More recent research has indicated that copper ions can bind within GRX’s active site unhindered by these prolines [15,16] Instead, other metallochaperones, such as ATOX1, with a higher metal affinity, outcompete GRX for metal cations [15]. Some proline-containing active site GRXs have been shown to form complexes to iron–sulfur clusters [17,18,19,20] suggesting that the notion that CPYC GRXs are always associated with redox chemistry while CGFS GRXs are always associated with metal atom/cluster transfer is an oversimplification [21]. As GRXs are integral proteins in the processing of intracellular ROS and contain the CPYC site, we suggest that silver can directly target GRX proteins during this silver-mediated ROS amplification process
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