This proposal aims at exploiting some of the striking and unexpected advances which were made in the past four years in our understanding of the structure and function of iron-sulfur (Fe-S) proteins. These advances were: discovery of 3Fe clusters (1979-80); recognition of these as [3Fe-4S] clusters (maintaining cubane-type structure) (1983); discovery of facile interconversions between [3Fe-4S] and [4Fe-4S] clusters in proteins (1981-82), formation of an alternative protein-bound linear cluster type, and most important, substrate binding to a specific cluster iron atom and participation of this cluster iron in an enzymatic reaction other than electron transfer. On this basis, it is planned to study with the Fe-S protein beef heart mitochondrial aconitase. The tools and methods will largely be standard ones in protein chemistry (PC), purification and analysis, and in metal and sulfide analysis in combination with the following spectroscopies: EPR, MB (Mossbauer), MCD, CD, RR (Resonance Raman), NMR, EXAFS and ENDOR. 1. Geometry of the active site via a) effects of tight binding substrates, analogues and inhibitors (MB, MCD, CD, RR, NMR, EPR); b) location of SH groups in the amino acid sequence (PC); and c) reagents specific for individual amino acids (PC). 2. Fe-S cluster substructure and magnetic coupling between metal ions via a) replacement of readily removable Fe by Zn2+, CO2+, Mn2+ or MO4+ (MB, MCD, EPR); b) S2- with Se2- or 32S2- by 34S2-; 56Fe by 57Fe or 54Fe(RR). 3. Reversible interconversions and oxido-reductions of various cluster forms and de novo cluster assembly via a) generation of linear [3Fe-4S] cluster and its reduction (EPR, MB, RR, PC); b) production of viable apoenzyme (PC); c) comparison of contact shifted cysteine protons with various enzyme forms (NMR). 4. Initial events in substrate and inhibitor binding (MB, MCD, RR) via a) rapid freeze-quenching and b) reaction in ethylene-glycol-H2O at -60C. 5. Essential general features of aconitases via comparative work on liver cytoplasmic aconitase or other enzymes catalyzing reactions, similar to those catalyzed by aconitase, which require Fe and thiol for activity. Basis of stability of [4Fe-4S] clusters in some proteins and lability in others with respect to decomposition to 3Fe cluster. 6. Development of methodology permitting use of the same sample for Mossbauer and EPR spectroscopy (as desirable in the work on aconitase) on the basis of the loop-gap resonator developed for EPR by Drs. Hyde and Froncisz at the National Biomedical ESR Center.
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