Mössbauer studies of beef heart aconitase: evidence for facile interconversions of iron-sulfur clusters.

Abstract

Beefheart aconitase, isolated under aerobic conditions, has been studied with Mossbauer and EPR spectroscopy. In the oxidized state, the enzyme exhibits an EPR signal at g = 2.01. The Mossbauer data show that this signal is associated with a 3Fe cluster. In dithionite-reduced aconitase, the 3Fe cluster, probably ofthe In the past few years, it has been recognized that many features ofFe-S proteins cannot be understood in terms ofthe structural types known for these proteins-i.e., FeS4, [2Fe-2S], [4Fe-4S], or 2[4Fe-4S].The occurrence of 3Fe clusters has been reported for a few proteins, of which ferredoxin I of Azotobacter vinelandii (1), ferredoxin II ofDesulfovibrio gigas (2), and beefheart aconitase (3) are probably the most intensively studied. X-ray diffraction studies on A. vinelandii ferredoxin (Fd) show that these clusters have a [3Fe-3S] core (4). The decisive spectroscopic evidence for the presence of a [3Fe-3S] center has been the observation ofan EPR signal at g = 2.01 in oxidized samples together with the unique Mossbauer spectra that these structures yield in the reduced state (5).Beef heart aconitase has properties not found in other proteins thought to contain [3Fe-3S] clusters. As obtained on routine purification, it is enzymatically inactive, but it can be reactivated by a number oftreatments; all ofwhich have in common the reduction of the Fe-S cluster (6, 7). Although inclusion of iron in the activation medium yields the highest activities, iron does not appear to be an obligatory ingredient of such media; activities up to 70% of maximum can be induced by some reducing agents alone (6, 7). We have now studied, by M6ssbauer spectroscopy, aconitase samples reduced and activated in a variety of ways. Only samples reduced and activated with dithionite (' 30% of maximal activity) have the Mossbauer features characteristic of [3Fe-3S] centers. Those activated with dithiothreitol/Fe2", dithionite/Fe2", or dithiothreitol alone display 60-100% activity and show entirely different Mossbauer features. We propose that the latter activation procedures convert the [3Fe-3S] cluster into a structure that has a core.In the present report, we limit ourselves to observations concerned with the process and consequences of reductive activation and oxidative deactivation of the enzyme. Conditions pertaining to the catalytic action of the enzyme will be considered elsewhere. MATERIALS AND METHODSAconitase was purified from beef heart as reported (8) with minor modifications. Analytical methods and EPR spectroscopy were as in ref. 9 and yielded results as in refs. 8 and 7 respectively. Activations were carried out anaerobically; for routine assays (10), 0.1 mM ferrous ethylene diammonium sulfate and 5 mM dithiothreitol were used, and Mbssbauer and EPR samples were prepared as desired for the specific purpose. Anaerobic procedures followed the outlines ofref. 11 with appropriate modifications.Although enzymatic assays for aconitase activity are simple and reproducible, it must be kept in mind that drawing ...

Further Characterization of the Spin Coupling Observed in Oxidized Hydrogenase from Chromatium vinosum. A Moessbauer and Multifrequency EPR Study

“…These observations are in accord with the published spin-coupling model (Papaefthymiou et al, 1987); namely, that the local spin of the Fe3+ site (S3 = 5 / 2 ) is antiparallel oriented to the system spin (S = 2) whereas the spin of the delocalized pair (Sl2 = 9 / 2 ) is aligned parallel to the system spin. The solid line in Figure l (Emptage et al, 1980;Huynh et al, 1980;Kent et al, 1982;Surerus et al, 1989). First, at temperature T 1 50 K the spectra consist of one well-defined quadrupole doublet with an isomer shift characteristic of high-spin ferric ions in a tetrahedral sulfur environment; the best studied protein, D.…”

“…This small value of J , however, is in severe conflict with SQUID magnetometry studies by Day et al (1988) and N M R studies by Macedo et al (1993) which favor J > 200 cm-l. The analyses of the low temperature Mossbauer spectra of a variety of [Fe&] l + ferredoxins have revealed substantial anisotropies of the 57Fe magnetic hyperfine interactions (Emptage et al, 1980;Huynh et al, 1980;Kent et al, 1982;Surerus et al, 1989). These observations are unusual for high-spin ferric ions but could be rationalized with the model of Bertrand et al (Gayda et al, 1982(Gayda et al, , 1983Bertrand et al, 1984;Guigliarelli et al, 1986a,b…”

A Developmentally Regulated Aconitase Related to Iron-regulatory Protein-1 Is Localized in the Cytoplasm and in the Mitochondrion of Trypanosoma brucei

“…Aconitase activity increased linearly with the amount of recombinant protein and was maximal between pH 7 and 8 (not shown). The activity was dependent on loading of the protein with Fe 2ϩ in the presence of DTT and was sensitive to oxygen (Table II), consistent with the presence of a labile [4Fe-4S] cluster (14,36,60). The conserved cysteine positions of TbACO ligating this putative iron-sulfur cluster (see Table I) were then substituted by serines by site-directed mutagenesis.…”

“…Activity of the enzyme critically depends on the presence of an iron sulfur [4Fe4S] cluster in the catalytic center that is highly sensitive to oxygen. Upon oxidation the cluster is converted to an inactive [3Fe4S] form (14). Mammalian cells express two distinct aconitases encoded by separate genes: (a) mitochondrial aconitase and (b) a bifunctional cytoplasmic aconitase that is identical to the iron-regulatory protein (IRP-1).…”

Nickel hydrogenases: in search of the active site

“…Some time later, inspired by the discovery of 3Fe clusters [59,111], Albracht et al [8] considered the possibility that the redox-induced changes might involve a 3Fe/4Fe cluster conversion. Subsequently it was recognized [9,10] vinosum strain DSM 185 {E~(pH 8.0) = + 150 mV [48].…”