ORIGINAL ARTICLE Ramesh B. Iyer Æ Radu Silaghi-Dumitrescu Donald M. Kurtz Jr Æ William N. Lanzilotta High-resolution crystal structures of Desulfovibrio vulgaris (Hildenborough) nigerythrin: facile, redox-dependent iron movement, domain interface variability, and peroxidase activity in the rubrerythrins Received: 11 February 2005 / Accepted: 13 April 2005 / Published online: 14 May 2005 Ó SBIC 2005 Abstract High-resolution crystal structures of Desulf- ovibrio vulgaris nigerythrin (DvNgr), a member of the rubrerythrin (Rbr) family, demonstrate an approxi- mately 2-A ˚ movement of one iron (Fe1) of the diiron site from a carboxylate to a histidine ligand upon conversion of the mixed-valent ([Fe2(II),Fe1(III)]) to diferrous states, even at cryogenic temperatures. This GluMHis ligand ‘‘toggling’’ of one iron, which also occurs in DvRbr, thus, appears to be a characteristic feature of Rbr-type diiron sites. Unique features of DvNgr re- vealed by these structures include redox-induced flipping of a peptide carbonyl that reversibly forms a hydrogen bond to the histidine ligand to Fe1 of the diiron site, an intra-subunit proximal orientation of the rubredoxin- (Rub)-like and diiron domains, and an electron transfer pathway consisting of six covalent and two hydrogen bonds connecting the Rub-like iron with Fe2 of the di- iron site. This pathway can account for DvNgr’s rela- tively rapid peroxidase turnover. The characteristic combination of iron sites together with the redox- dependent iron toggling between protein ligands can account for the selectivity of Rbrs for hydrogen peroxide over dioxygen. Keywords Non-heme diiron peroxidase Æ Rubrerythrin Æ Rubredoxin Æ Nigerythrin Æ Electron transfer Abbreviations Rbr: rubrerythrin Æ Rub: rubredoxin Æ Ngr: nigerythrin Æ Dv: Desulfovibrio vulgaris Æ ENDOR: Electron-nuclear double resonance Æ ZnS 4 Rbr: Rbr with zinc in the Rub-like site Æ PEG: polyethylene glycol Introduction Rubrerythrin (Rbr) is a homodimeric protein, each subunit of which consists of a four-helix bundle do- main containing a non-heme, non-sulfur diiron site and a rubredoxin-(Rub)-like domain containing a mononuclear [Fe(SCys) 4 ] site [1, 2]. Genomic analyses have shown that Rbr-like proteins are widespread in archaea and bacteria, particularly in air-sensitive microorganisms, and genomes of many anaerobes en- code multiple Rbr homologues [3–5]. A protein dub- bed ‘‘sulerythrin’’ has recently been reported from a strictly aerobic archaeon [6, 7]. Sulerythrin contains the four-helix bundle domain and diiron site ligand resi- dues that are characteristic of the Rbr family but lacks the Rub-like domain. The genome for this archaeon does not encode any Rub or Rub-like protein. More- over, no enzymatic activity or function has been re- ported for sulerythrin. The function of Rbr has been disputed since its first reported isolation from the anaerobic sulfate-reducing bacterium, Desulfovibrio vulgaris (Dv) in 1988 [8]. Re- cent evidence, both in vivo [4, 9–11] and in vitro [3, 12– 14], however, strongly supports a role in a novel oxi- dative stress protection system, in which Rbr functions as the terminal component of a NAD(P)H-dependent Electronic Supplementary Material Supplementary material is available for this article at http://dx.doi.org/10.1007/s00775-005- 0650-8. Ramesh B. Iyer and Radu Silaghi-Dumitrescu contributed equally to this work. R. B. Iyer Æ R. Silaghi-Dumitrescu Æ D. M. Kurtz Jr Department of Chemistry, University of Georgia, Athens, GA 30602, USA W. N. Lanzilotta Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA R. B. Iyer Æ R. Silaghi-Dumitrescu Æ D. M. Kurtz Jr W. N. Lanzilotta (&) Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602, USA E-mail: wlanzilo@bmb.uga.edu J Biol Inorg Chem (2005) 10: 407–416 DOI 10.1007/s00775-005-0650-8