In this work on aconitase focus will be on three main problem areas of general interest: 1) Fe-S cluster 3D and electronic structure and substructure. 2) Fe-S clusters as catalysts in reactions other than net e transfer. 3) Generality of point 2) above, i.e., enzymes of similar properties. The principal techniques (other than enzyme chemistry) will be mentioned in brackets using these abbreviations: Mossbauer, MB; Resonance Raman RR; Protein Chemistry, PC; with CD,MCD,EPR,NMR,ENDOR,EXAFS considered standard abbreviations. Comments to: 1. a) Spin coupling in Fe-S clusters, cluster (electronic) substructure, i.e. development of localized valence states in spin coupled clusters (MB, EPR, cluster ENDOR, (57 Fe, 33 S, 77 Se); RR (57, 54 Fe, 34 S, 80 Se)). b) Replacement of cluster metal components with other metal ions such as (Zn2+, Cd2+, Co2+, Ga3+, Cu+)(MB, EPR, ENDOR, RR) c) From a structural viewpoint: attachment to cluster of ligands, substrates, inhibitors (is there expansion of coordination sphere or replacement of existing ligands?) (MB, EPR, ligand or cluster ENDOR (57 Fe, 33 S, 1 H, 2 H, 13 C, 17 O), RR (34 S, 18 O, 57 Fe)) d) Cluster interconversions (MB, ENDOR, EPR, RR) and oxidation states of different forms, stability, intermediates and breakdown products. e) SH-chemistry of protein and apoprotein and relation to cluster structure (PC) 2. a) From a functional viewpoint (cf. 1c): cluster- substrate interactions. Rapid-freeze quenching to monitor initial events with various substrates or analogs (MB, ENDOR, EPR, RR). b) Special substrates or analogs, e.g. (+) or (-) erythrofluorocitrate (what is the reaction product?), fluorodeoxycitrate, oxalomalate. 3. Enzymes of properties similar to aconitase, i.e. Fe 2+ and RSH activated, unstable in air (do they represent a new class of Fe-S enzymes specializing in catalysis rather than net e-transfer between proteins, or cofactors?) (MB, EPR, cluster and ligand ENDOR, RR, possibly NMR, CD, MCD and EXAFS).
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