This is a proposal for support of work on the role of nitric oxide and cytosolic aconitase (aka iron regulatory protein, irp) in iron metabolism. In mammalian cells there are two forms of aconitase. The mitochondrial form (m-acon) participates in energy metabolism as part of the Krebs cycle. This is a fairly well understood enzyme that has been studied for many years by, among others, Helmut Beinert and Dr. Kennedy and C. D. Stout who determined its 3-dimensional structure. An exciting event in the regulation of iron metabolism was the finding that the cytosolic form of aconitase(c-acon) was, in fact, identical with IRP. Sequence analyses and other considerations led to the hypothesis that the two aconitases must have very similar structures even while possessing markedly different biological functions. Thus, in iron deficit, IRP binds to stem-loop structures (IREs) in ferritin mRNA and transferrin receptor mRNA. Translation of ferritin mRNA is suppressed while the life-time of the TrR-mRNA is prolonged leading to increased amounts of apo-transferrin. In iron surfeit, c-acon apparently binds one or more Fe and no longer binds to the IREs resulting in a reversal of the above described protein expression. 1.) Pure c-acon, isolated from iron sufficient cells, contains a single [4Fe-4S] cluster and one additional Fe, presumably as an isolated Fe(II) ion. 2.) That NO reacts with m-acon to destroy its aconitase acitvity. 3.) That NO reacts with m-acon to form compounds having EPR properties characteristic of Fe(NO)2. This proposal is focussed primarily on the c-acon and the role of iron and NO in mediating its interaction with RNA. The working hypotheses appear to be that the non-Fe/S iron plays an important role in mediating the protein-mRNA interaction and endogenous NO plays a role in this process. There are 3 Specific Aims: 1.) The Fex will be specifically labeled first with 59Fe then later with 57Fe. Different forms of the labeled c-acon or IRP will be examined spectroscopically using UV-Vis, EPR and Mossbauer spectroscopies. 2.) A detailed study of the reaction of NO, peroxynitrite and nitroxyl with the 3Fe and 4Fe forms of both m-acon and c-acon will be carried out. The products of the reactions will be characterized chemically and spectroscopically and will be tested for their ability to re-form active enzymes and, in the case of IRP, to bind the specific RNA sequences of the IREs. 3.) In several collaborative studies, Dr. Kennedy will continue to collaborate with Dr. D. Stout on crystallization experiments with; Dr. R. Eisenstein with IRP for examination of protein phosphorylation; and with Dr. L. Kiessling who will carry out cross-linking and binding experiments of IRP with IREs.
