BIOINFORMATICS<-->STRUCTURE
Jerusalem, Israel, November 17-21, 1996

Abstract


Three dimensional structure of HiPIP II isolated from Ectothiorhodospira halophila at 1.7 Å resolution from a twinned monoclinic crystal

C. Fraz�o (1), I. Bento (1), L. Sieker(2), F. Capozzi(3), C. Luchinat(3), Z. Dauter (4), K. Wilson (4), R. Herbst-Irmer (5), G. M. Sheldrick (5) and M. A. Carrondo (1)

(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

carrondo@ctqb01.itqb.unl.pt


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.

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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.


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