We report in this paper that highly purified Escherichia coli dihydroxy-acid dehydratase, fumarase A, fumarase B, and mammalian aconitase are inactivated by O2- with second order rate constants in ...the range of 10(6) to 10(7) M-1 s-1. Each of these enzymes belongs to the hydro-lyase class and contains catalytically active 4Fe-4S clusters. Simultaneous with inactivation by O2- is the release of iron from their clusters. Our working hypothesis is O2- inactivates these enzymes by oxidizing their clusters to an unstable oxidation state, and cluster degradation follows. Consistent with this hypothesis is our observation that spinach dihydroxy-acid dehydratase, a member of the hydro-lyase class that has a catalytically active 2Fe-2S cluster, is not inactivated and does not lose iron in the presence of O2-. Porcine fumarase, a member of the hydro-lyase class that does not contain an Fe-S cluster, is also not inactivated by O2-. We also report the rate constants for the inactivation of E. coli dihydroxy-acid dehydratase, fumarase A, fumarase B, and mammalian aconitase by O2 are close to 2 x 10(2) M-1 s-1, and the rate constants for the inactivation of E. coli dihydroxy-acid dehydratase and mammalian aconitase by H2O2 are about 10(3) M-1 s-1. E. coli dihydroxy-acid dehydratase has been reported previously to be inactivated in vivo when cells are grown in hyperbaric O2, presumably due to the increased O2- generated under these conditions. We report here that E. coli fumarase A, fumarase B, and aconitase are also inactivated in vivo by hyperbaric O2. Thermodynamic parameters for the oxidation of the cluster of aconitase by O2- and O2 are calculated.
It has been known for many years that fluoroacetate and fluorocitrate when metabolized are highly toxic, and that at least one effect of fluorocitrate is to inactivate aconitase. In this paper we ...present evidence supporting the hypothesis that the (-)-erythro diastereomer of 2-fluorocitrate acts as a mechanism based inhibitor of aconitase by first being converted to fluoro-cis-aconitate, followed by addition of hydroxide and with loss of fluoride to form 4-hydroxy-trans-aconitate (HTn), which binds very tightly, but not covalently, to the enzyme. Formation of HTn by these reactions is in accord with the working model for the enzyme mechanism. That HTn is the product of fluorocitrate inhibition is supported by the crystal structure of the enzyme-inhibitor complex at 2.05- angstrom resolution, release of fluoride stoichiometric with total enzyme when (-)-erythro-2-fluorocitrate is added, HPLC analysis of the product, slow displacement of HTn by 106-fold excess of isocitrate, and previously published Mossbauer experiments. When (+)-erythro-2-fluorocitrate is added to aconitase, the release of fluoride is stoichiometric with total substrate added, and HPLC analysis of the products indicates the formation of oxalosuccinate, and its derivative α -ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (-)-erythro-2-fluorocitrate. The structure of the inhibited complex reveals that HTn binds like the inhibitor trans-aconitate while providing all the interactions of the natural substrate, isocitrate. The structure exhibits four hydrogen bonds <2.7 angstrom in length involving HTn, H2O bound to the 4Fe-4S cluster, Asp-165 and His-167, as well as low temperature factors for these moieties, consistent with the observed very tight binding of the inhibitor.
Dihydroxy-acid dehydratase has been purified from Escherichia coli and characterized as a homodimer with a subunit molecular
weight of 66,000. The combination of UV visible absorption, EPR, magnetic ...circular dichroism, and resonance Raman spectroscopies
indicates that the native enzyme contains a 4Fe-4S2+,+ cluster, in contrast to spinach dihydroxy-acid dehydratase which
contains a 2Fe-2S2+,+ cluster (Flint, D. H., and Emptage, M. H. (1988) J. Biol. Chem. 263, 3558-3564). In frozen solution,
the reduced 4Fe-4S+ cluster has a S = 3/2 ground state with minor contributions from forms with S = 1/2 and possibly S =
5/2 ground states. Resonance Raman studies of the 4Fe-4S2+ cluster in E. coli dihydroxy-acid dehydratase indicate non-cysteinyl
coordination of a specific iron, which suggests that it is likely to be directly involved in catalysis as is the case with
aconitase (Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H., and Münck, E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80,
4674-4678). Dihydroxy-acid dehydratase from E. coli is inactivated by O2 in vitro and in vivo as a result of oxidative degradation
of the 4Fe-4Scluster. Compared to aconitase, the oxidized cluster of E. coli dihydroxy-acid dehydratase appears to be less
stable as either a cubic or linear 3Fe-4S cluster or a 2Fe-2S cluster. Oxidative degradation appears to lead to a complete
breakdown of the Fe-S cluster, and the resulting protein cannot be reactivated with Fe2+ and thiol reducing agents.
Iron(III) binding to the DOPA-containing Mytilus edulis adhesive protein (Mefp1) has been studied by spectrophotometric titrations, electron paramagnetic resonance (EPR), and resonance Raman ...spectroscopies. At pH 7.0, two different forms of the iron−protein complex exist: one purple (λmax = 548 nm) and one pink (λmax = 500 nm). The pink form is favored at high DOPA:Fe ratios and the purple at low DOPA:Fe ratios. Resonance Raman spectroscopy of both forms demonstrates that the chromophores are ferric catecholate complexes. EPR spectra of both forms of the protein measured at the same iron concentration reveal a g ≈ 4.3 resonance of approximately 4 times the intensity in the spectrum of the pink complex compared with that of the purple form. On the basis of the collective evidence obtained here, a model for the purple form of the ferric Mefp1 involving bis(catecholato) coordination of ferric ions with most of the iron(III) complexed as EPR-silent μ-oxo- or μ-hydroxo-bridged binuclear clusters is suggested. In the pink form, in contrast, the ferric iron is EPR-active, mononuclear, and present in high-spin tris(catecholato) complexes. The biological implications of these complexes are discussed.
It has been shown previously that Escherichia coli contains three fumarase genes designated fumA, fumB, and fumC. The gene products fumarases A, B, and C have been divided into two classes. Class I ...contains fumarases A and B, which have amino acid sequences that are 90% identical to each other, but have almost no similarity to the sequence of porcine fumarase. Class II contains fumarase C and porcine fumarase, which have amino acid sequences 60% identical to each other Woods, S.A., Schwartzbach, S.D., & Guest, J.R. (1988) Biochim. Biophys. Acta 954, 14-26. In this work it is shown that purified fumarase A contains a 4Fe-4S cluster. This conclusion is based on the following observations. Fumarase A contains 4 Fe and 4 S2- per mole of protein monomer. (The mobility of fumarase A in native polyacrylamide gel electrophoresis and the elution volume on a gel permeation column indicate that it is a homodimer.) Its visible and circular dichroism spectra are characteristic of proteins containing an Fe-S cluster. Fumarase A can be reduced to an EPR active-state exhibiting a spectrum consisting of a rhombic spectrum at high fields (g-values = 2.03, 1.94, and 1.88) and a broad peak at g = 5.4. Upon addition of substrate, the high field signal shifts upfield (g-values = 2.035, 1.92, and 1.815) and increases in total spins by 8-fold, while the g = 5.4 signal disappears.
Methods are described for the convenient preparation of aconitase from beef heart mitochondria in its inactive 3Fe-4S form and largely in its active 4Fe-4S form. Inactive aconitase can be activated ...anaerobically by various reducing agents without addition of iron. Under these conditions, maximally 70-80% of the activity attainable in the presence of added iron can be reached. It is concluded that during activation without added iron, 4Fe-4S clusters are built from 3Fe-4S clusters at the expense of a fraction of the 3Fe clusters present. This explains the approximately 75% maximal activation observed and concomitant loss of approximately 25% of total clusters as quantitated by EPR. Time course plots of aconitase activation appear to be second order but are not amenable to simple kinetic analysis because of the requirements of both reduction and Fe2+ for activation. Activation of aconitase with 59Fe leads to rapid (minutes) incorporation of 1 iron atom/cluster, which on subsequent inactivation is readily lost again. With longer incubation times (hour), 59Fe is found in more than a single site/cluster. It is concluded that, in analogy to cluster loss during activation in absence of added iron, the appearance of 59Fe in more than one cluster site can be due to complete breakdown and rebuilding of clusters. However, exchange into intact clusters cannot be ruled out. Ferric iron can be bound nonspecifically to active and inactive aconitase but can be readily removed by chelating agents. Sulfide is not required for activation of aconitase in keeping with the proposal that inactive aconitase, as isolated, contains a 3Fe-4S cluster. It is demonstrated that oxidation initiates the inactivation of aconitase with concomitant release of iron and formation of 3Fe clusters as determined by EPR.
Dihydroxy acid dehydratase, the third enzyme in the branched-chain amino acid biosynthetic pathway, has been purified to homogeneity (5000-fold) from spinach leaves. The molecular weights of ...dihydroxy acid dehydratase as determined by sodium dodecyl sulfate and native gel electrophoresis are 63,000 and 110,000, respectively, suggesting the native enzyme is a dimer. 2 moles of iron were found per mol of protein monomer. Chemical analyses of iron and labile sulfide gave an Fe/S2- ratio of 0.95. The EPR spectrum of dithionite-reduced enzyme (gavg = 1.91) is similar to spectra characteristic of Rieske Fe-S proteins and has a spin concentration of 1 spin/1.9 irons. These results strongly suggest that dihydroxy acid dehydratase contains a 2Fe-2S cluster, a novel finding for enzymes of the hydrolyase class. In contrast to the Rieske Fe-S proteins, the redox potential of the Fe-S cluster is quite low (-470 mV). Upon addition of substrate, the EPR signal of the reduced enzyme changes to one typical of 2Fe ferredoxins (gavg = 1.95), and the visible absorption spectrum of the native enzyme shows substantial changes between 400 and 600 nm. Reduction of the Fe-S cluster decreases the enzyme activity by 6-fold under Vmax conditions. These results suggest the direct involvement of the 2Fe-2S cluster of dihydroxy acid dehydratase in catalysis. Similar conclusions have been reached for the catalytic involvement of the 4Fe-4S cluster of the hydrolyase aconitase (Emptage, M. H., Kent, T. A., Kennedy, M. C., Beinert, H., and Münck, E. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4674-4678).
Endonuclease III is an iron-sulfur protein Cunningham, Richard P; Asahara, Hitomi; Bank, Janet F ...
Biochemistry (Easton),
05/1989, Letnik:
28, Številka:
10
Journal Article
Recenzirano
Elemental analyses, Mössbauer, and EPR data are reported to show that endonuclease III of Escherichia coli is an iron-sulfur protein. Mössbauer spectra of protein freshly prepared from E. coli grown ...on 57Fe-enriched medium demonstrate that the native enzyme contains a single 4Fe-4S cluster in the 2+ oxidation state, with a net spin of zero. Upon treatment with ferricyanide, a fraction (less than 25%) of the clusters is oxidized into a state which yields an EPR spectrum near g = 2.01 typical of a 3Fe-4S cluster. The magnetic field dependence of the linear electric field effect verifies this assignment. Electron spin echo modulation on the g = 2.01 form of the protein in deuterated solvent indicates the presence of exchangeable protons in the vicinity of the 3Fe-4S cluster. The data obtained show that the 4Fe-4S2+ cluster of the native enzyme is resistant to either oxidation or reduction, although photoreduction elicited a g = 1.94 type EPR signal characteristic of a 4Fe-4S1+ cluster. These studies show that endonuclease III is unique in being both a DNA repair enzyme and an iron-sulfur protein. The function of the 4Fe-4S cluster remains to be established.
Beef heart 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 of the 3Fe-3S type, is in a paramagnetic state of integer electronic spin (S = 2); the Mossbauer spectra exhibit all the unique features reported for proteins with 3Fe clusters. On activation of aconitase with ferrous ion, the paramagnetic 3Fe cluster of dithionite-reduced enzyme is converted into a diamagnetic (S = 0) form. Activation studies with iron enriched in either57Fe or56Fe suggest that activation transforms the 3Fe cluster into a center that has a 4Fe-4S core. This conclusion is supported by the observation that EPR signals characteristic of reduced 4Fe-4S clusters can be elicited under appropriate conditions. It has frequently been assumed that the activation of aconitase with Fe2+produces an active site containing a single ferrous ion. The data reported here suggest that a ferrous ion is used to rebuild a 4Fe-4S cluster.
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