Reversible Sequestration of Active Site Cysteines in a 2Fe-2S-bridged Dimer Provides a Mechanism for Glutaredoxin 2 Regulation in Human Mitochondria

Abstract

Human mitochondrial glutaredoxin 2 (GLRX2), which controls intracellular redox balance and apoptosis, exists in a dynamic equilibrium of enzymatically active monomers and quiescent dimers. Crystal structures of both monomeric and dimeric forms of human GLRX2 reveal a distinct glutathione binding mode and show a 2Fe-2S-bridged dimer. The iron-sulfur cluster is coordinated through the N-terminal active site cysteine, Cys-37, and reduced glutathione. The structures indicate that the enzyme can be inhibited by a high GSH/GSSG ratio either by forming a 2Fe-2S-bridged dimer that locks away the N-terminal active site cysteine or by binding non-covalently and blocking the active site as seen in the monomer. The properties that permit GLRX2, and not other glutaredoxins, to form an iron-sulfur-containing dimer are likely due to the proline-toserine substitution in the active site motif, allowing the main chain more flexibility in this area and providing polar interaction with the stabilizing glutathione. This appears to be a novel use of an iron-sulfur cluster in which binding of the cluster inactivates the protein by sequestering active site residues and where loss of the cluster through changes in subcellular redox status creates a catalytically active protein. Under oxidizing conditions, the dimers would readily separate into iron-free active monomers, providing a structural explanation for glutaredoxin activation under oxidative stress.

Arabidopsis Chloroplastic Glutaredoxin C5 as a Model to Explore Molecular Determinants for Iron-Sulfur Cluster Binding into Glutaredoxins

“…The rest of the new GSH/protein interactions are quaternary in nature, involving hydrogen bond donors or acceptors of the 28 WCSYC 32 signature of monomer B for the GSH molecule of monomer A. These interactions are similar to those observed in the crystal structure of the holoform of hGrx2 (25).…”

“…Overall, this indicates that AtGrxC5 likely functions through a monothiol mechanism involving only Cys 29 , but it does not clarify the role of Cys 32 . A comparison between structures of reduced and glutathionylated forms of PtGrxC1 and human Grx2 (although GSH is not covalently linked in this case), which are both dithiol Grxs, shows that thiol groups of the active site cysteines do not adopt the same conformers in both forms (8,24,25). Whereas the cysteines are distant and not oriented in the same direction in the reduced forms, the thiol groups of both active site cysteines come closer upon glutathionylation, which might allow formation of an intramolecular bridge if no other unfavorable factor exists.…”

“…7). The two residues Tyr 30 and Ser 31 also present in the signature of human (hGrx2) undergo the same conformational rearrangement upon Fe-S cluster binding in both enzymes (Table 3) (25). Gln 62 is the last residue that attracted our attention.…”

“…Both lateral chains exhibit different rotamers (Table 3). In GrxC5 apo , the thiol group of Cys 32 is in the vicinity of the catalytic cysteine (Cys 29 ) and interacts with the backbone NH group of Lys 26 , whereas in PtGrxS12, the side chain of Ser 32 points toward the hydroxyl group of Ser 25 .…”

“…The determination of the three-dimensional structure of poplar GrxC1 and human Grx2 indicated that the [2Fe-2S] cluster was ligated into a homodimer by the N-terminal active site cysteine of two monomers and two glutathione molecules (8,24,25). A similar ligation was observed for E. coli Grx4, a class II Grx, but the different orientation of the two monomers was proposed to be responsible for the differential cluster lability (26).…”