JBIC (1997) 2 : 680–689 Q SBIC 1997 ORIGINAL ARTICLE Ana V. Coelho 7 Pedro Matias 7 Vilmos Fülöp Andrew Thompson 7 Ana Gonzalez Maria A. Carrondo Desulfoferrodoxin structure determined by MAD phasing and refinement to 1.9-Å resolution reveals a unique combination of a tetrahedral FeS 4 centre with a square pyramidal FeSN 4 centre Received: 16 April 1997 / Accepted: 31 July 1997 A.V. Coelho Chemistry Department, Universidade de Évora, P-7000 Évora, Portugal A. Thompson EMBL Grenoble Outstation, c/o ILL 20, BP-156, F-38042 Grenoble Cedex, France A. Gonzalez 1 ESRF, BP-220, F-38043 Grenoble Cedex, France V. Fülöp Laboratory of Molecular Biophysics and Oxford Centre for Molecular Sciences, University of Oxford, South Parks Road, Oxford OX1 3QU, UK A.V. Coelho 7 P. Matias 7 M.A. Carrondo (Y) Instituto de Tecnologia Química e Biológica, Apartado 127, P-2780 Oeiras, Portugal Tel.: c351-1-4418215; Fax: c351-1-4411277; e-mail: carrondo6itqb.unl.pt Present address: 1 EMBL, c/o DESY, Notkestrasse 85, D-22603 Hamburg, Germany Abstract The structure of desulfoferrodoxin (DFX), a protein containing two mononuclear non-heme iron centres, has been solved by the MAD method using phases determined at 2.8 Å resolution. The iron atoms in the native protein were used as the anomalous scat- terers. The model was built from an electron density map obtained after density modification and refined against data collected at 1.9 Å. Desulfoferrodoxin is a homodimer which can be described in terms of two do- mains, each with two crystallographically equivalent non-heme mononuclear iron centres. Domain I is simi- lar to desulforedoxin with distorted rubredoxin-type centres, and domain II has iron centres with square py- ramidal coordination to four nitrogens from histidines as the equatorial ligands and one sulfur from a cysteine as the axial ligand. Domain I in DFX shows a remarka- ble structural fit with the DX homodimer. Further- more, three b-sheets extending from one monomer to another in DFX, two in domain I and one in domain II, strongly support the assumption of DFX as a functional dimer. A calcium ion, indispensable in the crystallisa- tion process, was assumed at the dimer interface and appears to contribute to dimer stabilisation. The C-ter- minal domain in the monomer has a topology fold sim- ilar to that of fibronectin III. Key words Desulfoferrodoxin 7 Non-heme iron centre 7 MAD method 7 Rubredoxin 7 Fibronectin III Abbreviations DFX desulfoferrodoxin 7 DX desulforedoxin 7 Rb rubredoxin 7 Rr rubrerythrin 7 Nl Neelaredoxin 7 D.d. Desulfovibrio desulfuricans ATCC 27774 7 D.v. Desulfovibrio vulgaris Hildenborough 7 D.g. Desulfovibrio gigas 7 rbo rubredoxin oxidoreductase Introduction The precise functional activity of several mononuclear non-heme iron proteins isolated from anaerobic sul- fate-reducing bacteria is still unknown [1–5]. However, these proteins seem to play a role in the electron trans- fer chain of the sulfate reducing-bacteria, and enzymat- ic functions can be attributed to some of them [5]. De- sulfoferrodoxin (DFX) has been isolated from the sul- fate-reducing bacteria Desulfovibrio (D.) desulfuricans ATCC 27774 (D.d.), grown in nitrate medium [1] and from D. vulgaris Hildenborough (D.v.), grown in sul- fate medium [1, 2]. Their molecular mass and subunit composition were determined by gel filtration and SDS-PAGE. Moura et al. found a 14 kDa monomer [1], while Verhagen et al. reported a dimer with a molecu- lar mass of 2!14 kDa [2]. A value of 13 881.3 Da was further determined by electrospray analysis for the pro- tein isolated from D. desulfuricans [6]. An iron content of two atoms per monomer was determined for both proteins. DFX was isolated from D. desulfuricans ATCC 27774 in two oxidation states; the oxidised, grey form and the semi-reduced, pink form. Both states can be in- terconverted by addition of appropriate oxidising or re-