Chapter 12
Structure and Environment of Metal Clusters in the Nitrogenase Molybdenum—Iron Protein from Clostridium pasteurianum 1
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Jeffrey T. Bolin , Nino Campobasso , Steven W. Muchmore , T. Vance Morgan , and Leonard E. Mortenson 2
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Downloaded by SUFFOLK UNIV on January 21, 2018 | http://pubs.acs.org Publication Date: July 26, 1993 | doi: 10.1021/bk-1993-0535.ch012
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Department of Biological Sciences, Purdue University, West Lafayette, IN 47907 Center for Metalloenzyme Studies, Department of Biochemistry, University of Georgia, Athens, GA 30602 The MoFe protein of Mo-dependent nitrogenases binds large and unusual metal-sulfur clusters of two types. These clusters are believed to be involved in the catalytic reduction of dinitrogen to ammonia by the enzyme. An analysis of the crystal structure of the MoFe protein from Clostridium pasteurianum at 2.2Åresolution is underway. On the basis of this analysis, atomic models for the structure of the FeMo cofactor and the Fe S cluster, the interactions of these clusters with the protein, and general features of the protein structure are described. Two possible through-bond paths for electron transfer the two clusters are identified. 8
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Nitrogenase catalyzes the reduction of dinitrogen to ammonia. The Mo-dependent form of the enzyme is a complex of two easily separated components known as the MoFe protein (component 1) and die Fe protein (component 2). MoFe protein is an (*2p2 tetramer, M = 220-230,000, which binds two Mo atoms and 30 Fe atoms in the form of four large metal-sulfur clusters of two types; two clusters of each type are present per tetramer. Fe protein is a homodimer, M = 60-70,000, which binds a single Fe4S4 cluster symmetrically between the subunits. The crystal structure of the Fe-protein has been described by Georgiadis et al (7). One of the metallic components of MoFe protein, the FeMo-cofactor (FeMoco), has been associated with substrate binding and reduction (2). Each FeMoco consists of one Mo, seven Fe, at least eight inorganic S atoms, plus one molecule of homocitrate. The two FeMoco units bound by the protein account for all of the Mo and 47% of the Fe content. Until recently, most models for the structure of MoFe protein assigned the remaining 16 Fe atoms to four atypical Fe4S4 clusters commonly referred to as "P-clusters." In 1987, Hagen et al (5) challenged the conventional model for the structure and redox chemistry of P-clusters and described possible alternatives, including the possibility of 8-Fe rather than 4-Fe clusters. Subsequently we reported X-ray anomalous diffraction experiments which conclusively demonstrated that MoFe protein binds four metal-sulfur clusters of roughly equivalent metal content, and that the non-cofactor Fe atoms are associated with two large FeS clusters of a previously undescribed type (4). We now know these clusters r
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0097-6156/93/0535-0186$06.00/0 © 1993 American Chemical Society
Stiefel et al.; Molybdenum Enzymes, Cofactors, and Model Systems ACS Symposium Series; American Chemical Society: Washington, DC, 1993.
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12. BOLIN ET AL.
Metal Clusters in ProteinfromC. pasteurianum
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arc identical, contain eight Fe atoms, and are constructed of two multiply-bridged 4Fe subclusters structurally similar to typical Fe4S4 clusters (see below). Within the past year, the crystal structures of A v l and Cpl, the MoFe proteins from Azotobacter vinelandii and Clostridium pasteurianum, respectively, have been determined to high resolution. Kim and Rees (5) published atomic models for both FeMoco and the 8-Fe cluster based on a 2.7Â resolution structure of A v l ; we will treat these initial "KR" models as reference structures for both clusters. We independently determined the structure of Cpl at 2.7Â resolution and subsequently extended this analysis to 2.2Â resolution. Here we describe structural features of Cpl based on this 2.2À resolution analysis with an emphasis on the structures of the clusters and their protein environments. We will compare these results with the KR models as well as with the results of our 2.7Â resolution analysis, both of which were presented orally at the symposium upon which this volume is based. Kim and Rees (6) revised their model for the 8-Fe cluster subsequent to the symposium and this revised model also will be considered. Procedures and Status of the C p l Model Monoclinic crystals of Cpl containing one
