6533b7d0fe1ef96bd125a4d3

RESEARCH PRODUCT

Stimulation of Fe-S cluster insertion into apoFNR by Escherichia coli glutaredoxins 1, 2 and 3 in vitro.

subject

description

Abstract The oxygen sensor fumarate nitrate reductase regu-lator (FNR) of Escherichia coli contains in the active (anaerobic)state a [4Fe–4S] 2þ cluster which is lost after exposure to O 2 .Inaerobically prepared apoFNR, or in FNR obtained by treatmentof [4Fe–4S] FNR with O 2 in vitro, intramolecular cysteinedisulfides are found, including the cysteine residues which serveas ligands for the Fe–S cluster. It is shown here that thereconstitution of [4Fe–4S] FNR from this form of aerobicapoFNR was preceded by a long lag phase when glutathione wasused as the reducing agent. Addition of E. coli glutaredoxins(Grx) 1, 2 or 3 decreased the lag phase greatly and stimulatedthe reconstitution rate slightly (about twofold). Reconstitution ofanaerobically prepared apoFNR, which has a lower cysteinedisulfide content, showed only a short lag phase, which furtherdecreased in the presence of Grx. It is concluded that in the lagphase the cysteine disulfides of apoFNR become reduced for theincorporation of the [4Fe–4S] cluster and that this reaction isstimulated by Grx. Thioredoxin (Trx) 1 showed no stimulationof FNR reconstitution in vitro. It is suggested that the function ofGrx might be of significance for the insertion of FeS cluster inproteins containing disulfides. 2004Federation ofEuropeanBiochemical Societies.Publishedby Elsevier B.V. All rights reserved.Keywords: Glutaredoxin; Thioredoxin; FNR; Oxygen sensor;Disulfide reductase; Glutathione1. IntroductionThe fumarate nitrate reductase regulator (FNR) of Esch-erichia coli controls the expression of a large number ofgenes in response to O