ABSTRACT
An increasing number of iron-sulfur (Fe-S) proteins are found in which the Fe-S cluster is not involved in net electron transfer, as it is in the majority of Fe-S proteins. Most of the former are (de)hydratases, of which the most extensively studied is aconitase. Approaches are described and discussed by which the Fe-S cluster of this enzyme could be brought into states of different structure, ligation, oxidation and isotope composition. The species, so obtained, provided the basis for spectroscopic and chemical investigations. Results from studies by protein chemistry, EPR, Mössbauer, 1H, 2H and 57Fe electron-nuclear double resonance spectroscopy are described. Conclusions, which bear on the electronic structure of the Fe-S cluster, enzyme-substrate interaction and the enzymatic mechanism, were derived from a synopsis of the recent work described here and of previous contributions from several laboratories. These conclusions are discussed and summarized in a final section.
Subject(s)
Aconitate Hydratase , Iron-Sulfur Proteins/analysis , Metalloproteins/analysis , Aconitate Hydratase/chemical synthesis , Aconitate Hydratase/metabolism , Apoenzymes/analysis , Apoenzymes/chemical synthesis , Apoenzymes/metabolism , Chemical Phenomena , Chemistry , Iron-Sulfur Proteins/chemical synthesis , Iron-Sulfur Proteins/metabolism , Isotope Labeling , Spectrum Analysis/methods , Substrate SpecificityABSTRACT
Apoascorbate oxidase has been shown to have a molecular weight of 137,000 +/- 3,000 and essentially the same gross quaternary conformation as native ascorbate oxidase. The apoenzyme, however, lacks much of the conformational stability of the native enzyme. The removal of the copper from the oxidase protein, and the simultaneous reduction of the disulfide bonds results in an apoenzyme of lower structural stability than the native oxidase. The aging of apoascorbate oxidase has been found to involve a loss of ionizable tyrosine residues and a dissociation to subunits and component polypeptide chains, which was not observed with the more stable native and holo enzymes. The molecular weight of holoascorbate oxidase has been determined to be 285,000. An s020, w of 9.79 has been determined for the holoenzyme. Holoascorbate oxidase has been shown to have an electrophoretic mobility on polyacrylamide gels that is 23% lower than either the native or apoenzyme. Furthermore, electrophoresis of the holoenzyme, in buffers containing dodecyl sulfate, and also isoelectric focusing of the holenzyme, produce patterns of greater similarity to those of apoascorbate oxidase than the native enzyme.
