Chloroplast monothiol glutaredoxins as scaffold proteins for the assembly and delivery of [2Fe–2S] clusters

Abstract

Glutaredoxins (Grxs) are small oxidoreductases that reduce disulphide bonds or protein-glutathione mixed disulphides. More than 30 distinct grx genes are expressed in higher plants, but little is currently known concerning their functional diversity. This study presents biochemical and spectroscopic evidence for incorporation of a [2Fe-2S] cluster in two heterologously expressed chloroplastic Grxs, GrxS14 and GrxS16, and in vitro cysteine desulphurase-mediated assembly of an identical [2Fe-2S] cluster in apo-GrxS14. These Grxs possess the same monothiol CGFS active site as yeast Grx5 and both were able to complement a yeast grx5 mutant defective in Fe-S cluster assembly. In vitro kinetic studies monitored by CD spectroscopy indicate that [2Fe-2S] clusters on GrxS14 are rapidly and quantitatively transferred to apo chloroplast ferredoxin. These data demonstrate that chloroplast CGFS Grxs have the potential to function as scaffold proteins for the assembly of [2Fe-2S] clusters that can be transferred intact to physiologically relevant acceptor proteins. Alternatively, they may function in the storage and/or delivery of preformed Fe-S clusters or in the regulation of the chloroplastic Fe-S cluster assembly machinery.

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

“…GrxS12, -S14, and -S16 in poplar as well as GrxS14 in A. thaliana (previously referred to as AtGRXcp) have already been confirmed as being located in plastids by GFP fusion (20,27,44). From localization prediction programs, only three additional A. thaliana Grxs exhibit a potential plastidial targeting sequence, namely AtGrxC5 (class I), AtGrxC10, and AtGrxS13 (class III) (10,11).…”

“…Iron concentrations were determined colorimetrically by using bathophenanthroline under reducing conditions after digestion of the protein in 0.8% KMnO 4 , 0.2 M HCl (35). Fe-S cluster reconstitution experiments in AtGrxC5 C32S were carried out under anaerobic conditions following a procedure described previously (20). UV-visible absorption and CD spectra were recorded at room temperature using a Shimadzu UV-3101PC spectrophotometer and Jasco J-715 spectropolarimeter, respectively.…”

“…Moreover, the close similarity of the spectroscopic properties of the [2Fe-2S] 2ϩ centers in AtGrxC5 and PtGrxC1 indicates analogous cluster coordination involving cysteinyl sulfur ligands from two GSHs and two Grxs. We have shown earlier by analyzing UVvisible absorption/CD and resonance Raman spectra that the Grx conformations are clearly distinct in class I and II Grxs, which both bind [2Fe-2S] clusters (8,20,26).…”

Structural and Spectroscopic Insights into BolA-Glutaredoxin Complexes

“…Plasmid Constructions-The cloning of AtBolA1 (At1g55805), AtBolA2 (At5g09830), BolA domain of AtSufE1 (At4g26500), AtGrxS14 (At3g54900), and AtGrxS17 (At4g04950) into pET vectors was described previously (10,12). For AtBolA1, another version coding a protein deprived of the first 63 amino acids instead of 51 amino acids was cloned into pET12a using 5Ј-CCCCCCCCCATATGGCAATAGAGAA-TCGA-3Ј (AtBolA1_for) and 5Ј-CCCGGATCCCTAGTCTT-TAGAAGGAGA-3Ј (AtBolA1_rev) primers to shorten the unstructured N-terminal part that likely prevented its crystallization.…”

“…Expression and Purification of Recombinant Proteins-Unlabeled tagged or untagged proteins were expressed and purified as previously (10,12 . For protein co-expression, an E. coli BL21(DE3) colony transformed by pCDFDuet HisAtGrxS14-BolA1 was grown at 37°C in LB media.…”