J. Mol. Biol. (1995) 251, 690–702 Crystal Structure of Desulforedoxin from Desulfovibrio gigas Determined at 1.8 Å Resolution: A Novel Non-heme Iron Protein Structure Margarida Archer 1 , Robert Huber 3 , Pedro Tavares 4 , Isabel Moura 4 Jose´ J. G. Moura 4 , Maria A. Carrondo 1,2 , Larry C. Sieker 5 , Jean LeGall 6 and Maria J. Roma˜o 1,2 * 1 Instituto de Tecnologia The crystal structure of desulforedoxin from Desulfovibrio gigas , a new Quı ´mica e Biolo´gica homo-dimeric (2×36 amino acids) non-heme iron protein, has been solved Rua da Quinta Grande by the SIRAS method using the indium-substituted protein as the single Apt. 127, 2780 Oeiras derivative. The structure was refined to a crystallographic R-factor of 16.9% at 1.8 Å resolution. Native desulforedoxin crystals were grown from either Portugal PEG 4K or lithium sulfate, with cell constants a = b = 42.18 Å, c = 72.22 Å 2 Instituto Superior Te´cnico (for crystals grown from PEG 4K), and they belong to space group P3 2 21. Departamento de Quı ´mica The indium-substituted protein crystallized isomorphously under the 1096 Lisboa Codex same conditions. Portugal The 2-fold symmetric dimer is firmly hydrogen bonded and folds as an incomplete b-barrel with the two iron centers placed on opposite poles of the 3 Max-Planck-Institut fu¨r molecule. Each iron atom is coordinated to four cysteinyl residues in a Biochemie, Martinsried distorted tetrahedral arrangement. Both iron atoms are 16 Å apart but Germany connected across the 2-fold axis by 14 covalent bonds along the polypeptide 4 Departamento de Quı ´mica chain plus two hydrogen bonds. (and Centro de Quı ´mica Fina Desulforedoxin and rubredoxin share some structural features but show e Biotecnologia), Faculdade de significant differences in terms of metal environment and water structure, Ciex ncias e Tecnologia which account for the known spectroscopic differences between rubredoxin Universidade Nova de Lisboa and desulforedoxin. 7 1995 Academic Press Limited 2825 Monte de Caparica Portugal 5 Department of Biological Structures, SM-20 University of Washington Seattle, WA 98195, USA 6 Department of Biochemistry and Molecular Biology University of Georgia Athens, GA 30602, USA Keywords: rubredoxin type proteins; desulforedoxin; sulfate reducing *Corresponding author bacteria; X-ray; 3D structure; crystallization Abbreviations used: Dx, desulforedoxin; Dfx, desulfoferrodoxin; DXLI, native crystals of Dx grown from lithium sulfate; DXPE, native crystals of Dx grown from PEG 4K; DXIN, derivative crystals of indium-substituted Dx grown from lithium sulfate; Rd, rubredoxin; r.m.s., root mean square; CSD, Cambridge Structural Database; SIRAS, single isomorphous replacement and anomalous scattering; PEG, polyethyleneglycol; EPR, electron paramagnetic resonance. Introduction Desulforedoxin (Dx), isolated from the sulfate reducing bacterium Desulfovibrio gigas , is a small metalloprotein (homodimer of molecular mass 7.9 kDa) containing an iron atom bound to four cysteine residues (Moura et al ., 1977), a metal center similar to the one found in rubredoxin (Rd). The latter protein has been extensively studied and its crystal structure analyzed at very high 0022–2836/95/350690–13 $12.00/0 7 1995 Academic Press Limited