An unusual mode of iron-sulfur-cluster coordination in a teleost glutaredoxin.

ABSTRACT

Glutaredoxins that contain a Cys-X-X-Cys active site motif are glutathione-dependent thiol-disulfide oxidoreductases. Vertebrate glutaredoxin 2 is characterized by two extra cysteines that form an intra-molecular disulfide bridge. Zebrafish glutaredoxin 2 contains four additional cysteines that are conserved within the infraclass of bony fish (teleosts). Here, we present a biochemical and biophysical characterization of zebrafish glutaredoxin 2, focusing on iron-sulfur-cluster coordination. The coordination of [2Fe2S](2+)-clusters in monomers of this protein was revealed by both absorption and Mössbauer spectroscopy as well as size exclusion chromatography. All other holo-glutaredoxins represent [FeS]-cluster bridged dimers using two molecules of non-covalently bound glutathione and the N-terminal active site cysteines as ligands. These cysteine residues were not required for [FeS]-cluster coordination in zebrafish glutaredoxin 2. A crystal structure of the teleost protein revealed high structural similarity to its human homologue. The two vertebrate-specific cysteines as well as two of the teleost-specific cysteines are positioned within a radius of 7Å near the C-terminus suggesting a potential role in [FeS]-cluster coordination. Indeed, mutated proteins lacking these teleost-specific cysteines lost the ability to bind the cofactor. Hence, the apparent mode of [FeS]-cluster coordination in zebrafish glutaredoxin 2 could be different from all yet described [FeS]-glutaredoxins.