NON-HEME IRON PROTEINS 417

H58 STRUCTURAL STUDIES ON A SIMPLE IRON-SULFUR PROTEIN DESULFOREDOXIN

Mauro Scharf', Carla S. Ascenso', Pedro Tavares', Brian J. GoodfeHow', Margarida Archer b, Jean LeGall*, Robert C. Scott', Frank Rusnak d, Maria A. Carrondo b, Robert Hnber', Maria J. Romfio b, Jos~ J. G. Moura" and Isabel Monra"

a-Dept.Quimica-FCT/UNL-PT, b-ITQB-UNL-PT, c-Dept.Biochem.Univ.Georgia/GA- USA, d-Dept Biochem. and Molec. Biology-Mayo Clinic FountL/Rochester-USA, e-Max-Planck Inst. far Biochem./Martinsried-Germany.

The most thoroughly characterized non-heme iron center in biology is Rubredoxin (Rd). Since it is the simplest member of the iron-sulfur class of metalloproteins, Rd serves as a prototype for protein-bound high-spin iron with tetrahedral coordination by sulfur atoms. The 3D structure is one of the most well resolved structures known. Desulforedoxin (Dx), isolated from the sulfate reducing bacterium Desulfovibrio (D.) gigas, is also a small protein (homo-dimer (2x36 aminoacids)) containing an iron atom bound by four cysteines, a metal center similar to the one found in Rd type proteins. This protein lacks histidine, arginine, proline, isoleucine, phenylalanine and tryptophan. Each monomer of Dx has one iron atom and four cysteines residues. The amino acid sequence of the polypeptide chain has an attractive structural feature due to the unusual sequence of the cystein¢ residues. Contrary to Rd type centers where the cystein pattern is:

--- Cys 9 --- X --- X --- Cys 12 --- [Xn] --- Cys 39--- Pro --- X --- Cys 42 --- Gly ---

Dx has the two final cysteines in consecutive positions: --- Cys 9 --- X --- X --- Cys 12 --- [Xn] --- Cys 28 --- Cys 29 --- Gly ---

It was speculated that the adjacent cystcines 28 and 29 impose some distortion on the tetrahedral coordination. The redox potential of the centre is -35 mV (in Rds 0/-50mV). The structural arrangement of the iron and the four cysteinyl ligands has been extensively probed using complementary methods. The conclusions drawn from the various approaches will be presented and include the following methodologies: O.V./Visible, EPR and M6ssbauer spectmscopies, chemical synthesis of the protein and metal site reoanstitution, X-ray crystal structure determination, solution structure determined by 2D NMR of the Zn-derivative, EXAFS, Electrochemistry and production of recombinant protein in E. coli. The results obtained by each type of approach will be discussed and it is concluded that the Fe site in Dx is coordinated by four cysteines, a suggestion that was previously done based on the amino acid sequence and spectroscopic data and now proved to be indeed correct. Support from JNICT-PRAXIS, EC Network ERBCHRCT920014. MS tanks CNPq/BraziL

References Moura, I., Tavares, P. & Ravi, N. in "Methods in Enzymology" (1994) vol.243,

Chap.15, eds. H. D. Peck, Jr and J. LeGall, Academic Press. Moura, I., Tavares, P., Moura, J.J.G., Ravi, N., Huynh, B.H., Liu, M.-Y. & LeGall,

J. (1990)J. Biol. Chem. 265, 21596-21602. Archer, M., Huber, R., Tavares, P., Moura, I., Moura, J.J.G:, Carrondo, M.A.,

Sieker, L.C., LeGall, J. & Rondo, M.J. (1995) J. Mol. Biol., submited. Tavares, P., Wunderlich, J. K., Loyal, S. G., LeGall, J., Moura, J. J. G., Moura, I.

(1995) Biochem.Biophys.Res.Commun. 208, 680-687.