(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, Università degli Studi di Bologna, Italy
(4) EMBL, c/o DESY, Hamburg, Germany
(5) Institut für
Anorganische Chemie der Universität Göttingen , 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 Mössbauer [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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