Functional, structural, and spectroscopic characterization of a glutathione-ligated [2Fe–2S] cluster in poplar glutaredoxin C1

Abstract

When expressed in Escherichia coli, cytosolic poplar glutaredoxin C1 (CGYC active site) exists as a dimeric iron-sulfur-containing holoprotein or as a monomeric apoprotein in solution. Analytical and spectroscopic studies of wild-type protein and site-directed variants and structural characterization of the holoprotein by using x-ray crystallography indicate that the holoprotein contains a subunit-bridging [2Fe-2S] cluster that is ligated by the catalytic cysteines of two glutaredoxins and the cysteines of two glutathiones. Mutagenesis data on a variety of poplar glutaredoxins suggest that the incorporation of an iron-sulfur cluster could be a general feature of plant glutaredoxins possessing a glycine adjacent to the catalytic cysteine. In light of these results, the possible involvement of plant glutaredoxins in oxidative stress sensing or ironsulfur biosynthesis is discussed with respect to their intracellular localization.iron-sulfur protein ͉ plant

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

“…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).…”

“…On the other hand, specific class I Grxs such as human Grx2 and plant GrxC1 also have the capacity to incorporate Fe-S clusters that are more stable than those found in class II Grxs (8,23). Based on the observation that oxidized glutathione promotes cluster disassembly in human Grx2, it has been hypothesized that under defined redox conditions the release of Grxs in an active form might serve as a redox sensor in human cells (23).…”

“…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). The capacity of class I Grxs to bind an Fe-S cluster has been studied in detail by site-directed mutagenesis, and it was proposed that the nature of the residues adjacent to the first active site Cys determined this capacity (8,27). In particular, for Grxs with a YCPFC or YCPYC active site, the replacement of the proline by a glycine, simulating GrxC1 (YCGYC), was sufficient to allow the incorporation of such a cluster (8) In addition, it has been hypothesized that the Trp residue adjacent to the catalytic cysteine residue in GrxS12 (WCSYS active site) prevented cluster incorporation because changing it into a Tyr allowed cluster incorporation (27).…”

“…This variation correlates with the type of Grx considered and depends on their redox potential and catalytic mechanism. Indeed, whereas classical Grxs have midpoint redox potentials between Ϫ170 and Ϫ230 mV (pH 7.0) (7,8), Chlamydomonas Grx3 forms during its catalytic cycle an atypical intramolecular disulfide between the first active site cysteine and a second cysteine located in the C-terminal part of the protein (6). The more negative redox potential (Ϫ323 mV at pH 7.9) observed for this protein most likely explains its ability to receive electrons from ferredoxin:thioredoxin reductase but not from glutathione (6).…”

Comparative analysis of glutaredoxin domains from bacterial opportunistic pathogens

“…This might kill bacteria by inhibition of DNA synthesis and/or through increases in ROS toxicity. Structures have been published for several forms of human (Sun et al, 1998;Yang et al, 1998), plant (Rouhier et al, 2007;Li et al, 2010), budding yeast (Gibson et al, 2008;Discola et al, 2009) and Escherichia coli GLXRs (Iwema et al, 2009;Fladvad et al, 2005;Xia et al, 1992Xia et al, , 2001Bushweller et al, 1994;Sodano et al, 1991). However, it was unclear whether other bacterial GLXRs would adopt similar conformations.…”