The state of cluster SH and S2- of aconitase during cluster interconversions and removal. A convenient preparation of apoenzyme.
During the interconversion of the various cluster types observed in aconitase ([4Fe-4S]1+,2+, [3Fe-4S]0,1+, cubane, or linear types) and production of apoenzyme, changes int he state of the sulfur ligands (RSH and S2-) are bound to occur. We have attempted to obtain information on such changes by interception of SH groups and/or by analysis of the resulting cluster or apoprotein for various forms of sulfur and enzymatic activity. During cluster interconversions no evidence was obtained for changes in SH titer that could be associated with cluster ligands. We conclude from this that the ligand exchange at the cluster is too rapid to allow trapping even by SH reagents of low Mr. The possibility that released SH becomes buried in the protein structure and unreactive cannot be entirely discounted. On formation of the apoenzyme by oxidation with ferricyanide, four SH groups per molecule became unavailable for reaction with 5,5'-dithiobis(2-nitrobenzoic acid); instead, while the holoenzyme contains no disulfides or S(0), two di- or polysulfides were found in the apoenzyme indicating that, on an average, four SH groups and three of the original four S(2-) ligands are trapped as RS-Sx-SR. In agreement with this conclusion is the fact that the apoenzyme can be reconstituted without addition of S(2-). A convenient preparative procedure for reconstitutable apoenzyme in 75% yield is [3Fe-4S] and [4Fe-4S] clusters with a variety of combinations of iron and sulfur isotopes as required for Mössbauer, resonance Raman, and electron nuclear double resonance studies.
Kennedy
M C
MC
Department of Biochemistry, Medical College of Wisconsin, Milwaukee 53226.
Beinert
H
H
D000154:Aconitate Hydratase; D001051:Apoenzymes; D001059:Apoproteins; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D007460:Iodoacetamide / Q000494:pharmacology; D007506:Iron-Sulfur Proteins; D008958:Models, Molecular
D001051:Apoenzymes; D001059:Apoproteins; D007506:Iron-Sulfur Proteins; D004228:Dithionitrobenzoic Acid; D000154:Aconitate Hydratase; D004229:Dithiothreitol; D007460:Iodoacetamide