A spin-label study of the disposition of the Fe-S cluster with respect to the active center of aconitase.
It has been reported by Johnson et al. ((1977) Biochem. Biophys. Res. Commun. 74, 384-389) that phenacyl bromide reacts with a single reactive sulfhydryl group of aconitase, abolishing enzyme activity. Substrate or analogs have a protective effect. This group is therefore at the catalytic site of the enzyme. Aconitase is also known to be an Fe-S protein, paramagnetic as obtained on purification (Ruzicka and Beinert (1978) J. Biol. Chem. 253, 2514-2517). We have attempted to obtain information on the location of the Fe-S cluster of aconitase with respect to the catalytically active site by attaching nitroxide-labelled sulfhydryl reagents of the bromoacyl and maleimide type to the sensitive sulfhydryl group. The EPR signals of those spin-labelled sulfhydryl reagents that abolish enzyme activity disappear during reaction with aconitase. EPR spectra at 13 K of the product obtained by reaction of three spin labels (two maleimides and one bromoacyl) with aconitase included a half-field transition at g approximately equal to 4.0 which is characteristic of spin-spin interaction. On the basis of calculations of the dependence of the intensity of the half-field transition on the distance between two interacting unpaired electrons (Eaton and Eaton, (1982) J. Am. Chem. Soc. 104, 5002-5003) the distances between the nitroxide N-O bond and the center of the Fe-S cluster for the three spin labels were calculated to be 10.5, 11 and 13 A. Combined distance and orientation data for the three spin labels indicate that the reactive sulfhydryl group is about 12 A from the center of the Fe-S cluster.
D000154:Aconitate Hydratase / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D007700:Kinetics; D008667:Metalloproteins / Q000378:metabolism*; D009206:Myocardium / Q000201:enzymology; D011487:Protein Conformation; D013113:Spin Labels / Q000138:chemical synthesis / Q000494:pharmacology*; D013329:Structure-Activity Relationship
D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; D013113:Spin Labels; D000154:Aconitate Hydratase