Mössbauer studies of aconitase. Substrate and inhibitor binding, reaction intermediates, and hyperfine interactions of reduced 3Fe and 4Fe clusters.
نویسندگان
چکیده
Active beef heart aconitase contains a [4Fe-4S] cluster. One iron of the cluster, Fea, is labile and can be removed easily by oxidation in air to yield the [3Fe-4S]1+ cluster of inactive aconitase. We have previously shown that substrate binds to Fea. We have continued our Mössbauer studies by further investigating the active and inactive forms of the enzyme. When active aconitase, [4Fe-4S]2+, is mixed with substrate, two species (substrates or intermediates bound to Fea) labeled S1 and S2 are obtained. With the nitroanalogs of citrate and isocitrate, thought to be transition state analogs, and fluorocitrate, species S2, but not S1, is observed, suggesting that S2 represents a carbanion transition state complex. We have prepared Mössbauer samples by rapid mix/rapid freeze techniques. Using either citrate, isocitrate or cis-aconitate, the natural substrates, we have been able to detect at 0 degree C reaction intermediates in the 5-35 ms time range and, studying enzyme substrate interactions at subzero temperatures in a water/methanol/ethylene glycol solvent, we have observed new species when substrates were added at -60 degrees C. Details of these experiments are given, although in neither case can unique interpretations be offered at this time. We have also investigated reduced active aconitase ([4Fe-4S]1+; EPR at g = 1.94) in the presence of substrate with material selectively enriched with 57Fe in either Fea or the other three cluster sites. The spectra were analyzed with a spin Hamiltonian, and the results are discussed and interpreted in terms of three inequivalent Fe sites in the cluster. Finally, we have studied enzyme containing the reduced [3Fe-4S]0 cluster. There is no indication that citrate binds to the 3Fe cluster, and since no significant activity was observed, we conclude that aconitase containing a 3Fe cluster is not active in either oxidation state.
منابع مشابه
Mössbauer studies of beef heart aconitase: evidence for facile interconversions of iron-sulfur clusters.
Beef heart aconitase, isolated under aerobic conditions, has been studied with Mössbauer and EPR spectroscopy. In the oxidized state, the enzyme exhibits an EPR signal at g = 2.01. The Mössbauer data show that this signal is associated with a 3Fe cluster. In dithionite-reduced aconitase, the 3Fe cluster, probably of the [3Fe-3S] type, is in a paramagnetic state of interger electronic spin (S = ...
متن کاملCharacterization of the Fe-S cluster in aconitase using low temperature magnetic circular dichroism spectroscopy.
Beef heart aconitase has been studied by low temperature magnetic circular dichroism (MCD) spectroscopy in the wavelength region 300 to 1900 nm. Together with parallel electron paramagnetic resonance and activity measurements, these data enable correlations between Fe-S cluster-type and enzymic activity in aconitase. In samples not exposed to extraneous Fe, the Fe-S cluster in aconitase exhibit...
متن کاملDirect square-wave voltammetry of superoxidized [4Fe-4S]3+ aconitase and associated 3Fe/4Fe cluster interconversions.
We report a direct square-wave voltammetric study of the iron-sulfur enzyme, aconitase, at the pyrolytic graphite edge electrode. New and established redox driven reactions were observed and the equilibrium reduction potential for each couple was determined: E0'[3Fe-4S]1+/0 = -268 mV, E0'[4Fe-4S]2+/1+ = -450 mV, E0'[4Fe-4S]3+/2+ = +100 mV, E0'Linear Form = -281 mV, and putatively, E0'[3Fe-4S]0/...
متن کاملOptical and EPR characterization of different species of active and inactive aconitase.
It has been shown by spectroscopic (Kent, T. A., Dreyer, J-L., Kennedy, M.C., Huynh, B.H., Emptage, M.H., Beinert, H., and Münck, E. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1096-1100) and chemical (Kennedy, M.C., Emptage, M.H., Dryer, J-L., and Beinert, H. (1983) J. Biol. Chem. 258, 11098-11105) methods that interconversion of [3Fe-4S] and [4Fe-4S] clusters underlies activation and inactivatio...
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ورودعنوان ژورنال:
- The Journal of biological chemistry
دوره 260 11 شماره
صفحات -
تاریخ انتشار 1985