(1) Instituto de Tecnologia Quimica e Biologica,
Rua da Quinta Grande 6,
Apartado 127,
2780 OEIRAS,
Portugal
(2) Department of Biological Structures, University of Washington, Seattle,USA
(3) Istituto di Chimica Agraria, Universit� degli Studi di Bologna, Italy
(4) EMBL, c/o DESY, Hamburg, Germany
(5) Institut f�r
Anorganische Chemie der Universit�t G�ttingen , Germany
The molecular structure of a high potential iron sulfur protein (HiPIP), the iso-II isozyme isolated from the halophilic purple bacterium Ectothiorhodospira halophila, has been solved by X- ray diffraction analysis to a resolution of 1.7 Å. HiPIP II and HiPIP I from the same source are the most acidic of the HiPIPs so far isolated, with net charges of -14 and -11 respectively [1]. Reduction potentials vary with pH and, at neutrality, Hipip II and I have the lower values for this class of proteins, 93 and 133mV, respectively [2]. NMR [3] and M�ssbauer [4] results indicate that in the oxidized form of Hipip II, the [4Fe-4S] cluster is formed by two iron (III) ions and one mixed- valence pair. This cluster is ligated to the protein via four cysteinyl sulfur ligands.
Synchrotron data were collected at the EMBL-Hamburg outstation. The 53819 reflexions were processed with DENZO/SCALEPACK [5] into 30130 unique intensities (92.1% completeness) with an Rmerge(I)=6.3%. The crystals belong to space group P21, a=28.5, b=122.2, c=45.7Å, beta=108.1 , which due to the singular cell dimensions relationship allow for a twinning by a two-fold rotation around axis a or axis c*. In fact, the metric symmetry appeared orthorhombic, but with a too high Rmerge. Evidence for twinning came from the Wilson distribution [6] and the too low <|E2-1|>=0.68. The HiPIP I structure [7] was used as search model for molecular replacement of the 4 molecules in the asymmetric unit; the solution could be consistently found with both programs AMoRe [8] and TFFC-CCP4 [9]. Non-crystallographic symmetry restraints have been applied in the HiPIP II twinned refinement, which has been carried out with program SHELXL-96 [10] (actual R=18% and Rfree=25% for an uncompleted refinement). The twin fraction is actually estimated as 0.47.
The X-ray structure of HiPIP II will be compared with that of other HiPIPs of known structures. A correlation was observed between the surface charges residues with the reduction potentials for a series of HiPIPs [2,11]. This structure determination will allow the assessment of the conclusions obtained based on a MD model [3] of HiPIP II. Furthermore, the existence of four independent molecules in the assymetric unit in the structure of HiPIP II will allow the calculation of a statistical average for each Fe-SCys distance. These values will bring more structural data to the discussion of the valence distribution localization within the cluster.
1- Meyer, T. E., Przysiecki, C. T., Watkins, J.A., Bhattacharyya, A.,
Simondsen, R. P.,
Cusanovich, M.A. and Tollin, G. (1983) Proc. Natl. Acad. Sci. U.S.A. 80,
6740-6744.
2- Luchinat, C., Capozzi, F., Borsari, M., Battistuzzi, G. and Sola, M.
Biochem.
Biophys. Res. Commun. (1994) 203,436-442.
3-Banci, L., Bertini, I., Capozzi, F., Carloni, P., Ciurli, S., Luchinat,
C., Piccioli, M., J.
Am. Chem. Soc, (1993), 115, 3431-3440.
4- Bertini, I., Campos, A.P., Luchinat, C., Teixeira, M., J. Inorg. Biochem.
(1993), 52,
227-234.
5- Gewirth, D., Otwinowski, Z. and Minor, W. (1995) The HKL Manual, Department
of Molecular Biophysics and Biochemistry, Yale University, U.S.A.
6- Intensity statistics: Rees, D.C. (1980) Acta Cryst. A36, 578-581.
7- Breiter, D. R., Meyer, T.E., Rayment, I., Holden, H. M. J. Biol. Chem.
(1991) 266,
18660-18667.
8- Navaza, J. (1994) Acta Cryst., Sect. A, 50, 157-163.
9- CCP4 (1979), The SERC (UK) Collaborative Computing Project No. 4, a Suite of
Programs for Protein Crystallography, distributed from Daresbury Laboratory,
Warrington WA4 4AD, UK.
10- Sheldrick, G.M. and Schneider, T.R.. (1996). SHELXL: High resolution
refinement.
Methods Enzymol., in the press.
11 - Banci, L. Bertini, Ciurli, S., Luchinat, C., Pierattelli, R., Inorg.
Chim. Acta, (1994)
240, 251-256.