Structure and Dynamics of Ferredoxins by NMR and X-ray

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Two-iron ((2Fe-2S)) ferredoxins comprise an important class of biological molecules whose function is incompletely understood at the molecular level. The investigators propose to study three classes of (2Fe-2S) ferredoxins: those found in mitochondria of steroid metabolizing tissue in vertebrates, those involved in photosynthesis, and those involved in nitrogen fixation. Examples of wild-type ferrodoxins from each of the three categories have been cloned and currently are overproduced in the PI's laboratory. The investigators previously determined x-ray structures from examples of two of the ferredoxin classes and an NMR structure from one. NMR and x-ray studies of members of the other classes of (2Fe-2S) ferredoxin are underway. The experiments are designed (1) to develop an understanding of factors that determine the reduction potential and electron delocalization in (2Fe-2S) ferredoxins, (2) to elucidate mechanisms of electron transfer to and from these ferredoxins, and (3) to investigate the requirements and energetics of (2Fe-2S) cluster formation. Roles of individual residues will be investigated by site-directed mutagenesis. Stable isotope labels inserted at specific locations will be used to monitor the electronic properties of atoms in the active site. X-ray structures will be determined for interesting mutant proteins that can by crystallized and analyzed either by multiple isomorphous replacement or by molecular replacement methods. %%% One of the basic motifs found in proteins that function as electron carriers is an iron atom coordinated by four sulfur atoms (an iron- sulfur cluster). Iron-sulfur clusters are found in all forms of life from bacteria to man. Iron-sulfur clusters can contain one, two, three, four, or more iron atoms, and their affinity for electrons varies over several orders of magnitude. They are involved in photosynthesis, respiration, nitrogen fixation, and many other important metabolic processes. This research project is designed to answer the following fundamental questions about proteins that contain iron-sulfur clusters: (1) How does the sequence of the protein specify the structure of the iron-sulfur cluster, the number of iron atoms and the geometry? (2) How does the sequence of the protein determine the affinity of the cluster for electrons and thus tune its reactivity for a particular process? (3) What residues in the protein are responsible for the specificity of electron transfer, and how do they guide that transfer? This knowledge will enable one to design electron transfer proteins with desired properties.