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J. Biol. Chem., Vol. 280, Issue 26, 24544-24552, July 1, 2005

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A Novel Monothiol Glutaredoxin (Grx4) from Escherichia coli Can Serve as a Substrate for Thioredoxin Reductase^*

Aristi Potamitou Fernandes, Malin Fladvad, Carsten Berndt, Cecilia Andrésen, Christopher Horst Lillig, Peter Neubauer¶, Maria Sunnerhagen, Arne Holmgren, and Alexios Vlamis-Gardikas||**

From the Medical Nobel Institute for Biochemistry, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden, the Department of Physics and Measurement Technology, Biology and Chemistry, Linköping University, Sweden, the ^¶Bioprocess Engineering Laboratory and Biocenter Oulu, Department of Process and Environmental Engineering, University of Oulu, FIN-90014 Oulu, Finland

Glutaredoxins are ubiquitous proteins that catalyze the reduction of disulfides via reduced glutathione (GSH). Escherichia coli has three glutaredoxins (Grx1, Grx2, and Grx3), all containing the classic dithiol active site CPYC. We report the cloning, expression, and characterization of a novel monothiol E. coli glutaredoxin, which we name glutaredoxin 4 (Grx4). The protein consists of 115 amino acids (12.7 kDa), has a monothiol (CGFS) potential active site and shows high sequence homology to the other monothiol glutaredoxins and especially to yeast Grx5. Experiments with gene knock-out techniques showed that the reading frame encoding Grx4 was essential. Grx4 was inactive as a GSH-disulfide oxidoreductase in a standard glutaredoxin assay with GSH and hydroxyethyl disulfide in a complete system with NADPH and glutathione reductase. An engineered CGFC active site mutant did not gain activity either. Grx4 in reduced form contained three thiols, and treatment with oxidized GSH resulted in glutathionylation and formation of a disulfide. Remarkably, this disulfide of Grx4 was a direct substrate for NADPH and E. coli thioredoxin reductase, whereas the mixed disulfide was reduced by Grx1. Reduced Grx4 showed the potential to transfer electrons to oxidized E. coli Grx1 and Grx3. Grx4 is highly abundant (750–2000 ng/mg of total soluble protein), as determined by a specific enzyme-link immunosorbent assay, and most likely regulated by guanosine 3',5'-tetraphosphate upon entry to stationary phase. Grx4 was highly elevated upon iron depletion, suggesting an iron-related function for the protein.

Received for publication, January 19, 2005 , and in revised form, April 4, 2005.

^* This work was supported by grants from the Wenner-Gren foundation, the Swedish Cancer Society, the Karolinska Institute, Linköping University, the Swedish Research Council, the Carl Trygger Foundation, the Swedish Society for Medical Research, the Knut and Alice Wallenberg Foundation, the TEKES Neobio program, and the Greek General Secretariat of Research and Technology in collaboration with the European Union (program Enter). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^|| Present address: Foundation of Biomedical Research, Academy of Athens, Soranou Efessiou 4, GR 11527 Athens, Greece.

^** To whom correspondence should be addressed: Medical Nobel Institute for Biochemistry, Dept. of Medical Biochemistry and Biophysics, Karolinska Institutet, S-171 77 Stockholm, Sweden, Tel.: 46-8-5248-7022; Fax: 46-8-305-193; E-mail: alexios.vlamis{at}mbb.ki.se.

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