Alternatives to petroleum-derived fuels are being sought in order to reduce the world's dependence on non-renewable resources. The most common renewable fuel today is ethanol derived from corn grain (starch) and sugar cane (sucrose). It is expected that there will be limits to the supply of these raw materials in the near future, therefore lignocellulosic biomass is seen as an attractive feedstock for future supplies of ethanol. However, there are technical and economical impediments to the development of a commercial processes utilizing biomass. Technologies are being developed that will allow cost-effective conversion of biomass into fuels and chemicals. These technologies include low-cost thermochemical pretreatment, highly effective cellulases and hemicellulases and efficient and robust fermentative microorganisms. Many advances have been made over the past few years that make commercialization more promising.

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.

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-4S]2+ 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-4S]1+ 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.

It has been known for many years that f luoroacetate and f luorocitrate when metabolized are highly toxic, and that at least one effect of f luorocitrate is to inactivate aconitase. In this paper we present evidence supporting the hypothesis that the (؊)-erythro diastereomer of 2-f luorocitrate acts as a mechanism based inhibitor of aconitase by first being converted to f luoro-cis-aconitate, followed by addition of hydroxide and with loss of f luoride 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 f luorocitrate inhibition is supported by the crystal structure of the enzymeinhibitor complex at 2.05-Å resolution, release of f luoride stoichiometric with total enzyme when (؊)-erythro-2-f luorocitrate is added, HPLC analysis of the product, slow displacement of HTn by 10 6 -fold excess of isocitrate, and previously published Mössbauer experiments. When (؉)-erythro-2-f luorocitrate is added to aconitase, the release of f luoride is stoichiometric with total substrate added, and HPLC analysis of the products indicates the formation of oxalosuccinate, and its derivative ␣-ketoglutarate. This is consistent with the proposed mechanism, as is the formation of HTn from (؊)-erythro-2-f luorocitrate. 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 Å in length involving HTn, H 2 O 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.The mechanism of the inhibitory effects of fluorocitrate on the enzyme aconitase [citrate(isocitrate)hydrolyase, EC 4.2.1.3] has been a long-standing problem in biochemistry. The toxic nature of fluoroacetate was discovered over 50 years ago (1, 2) and citrate was found to accumulate in tissues poisoned with compounds that could provide the fluoroacetyl residue. On this basis Peters (3) and Martius (4) proposed that the inhibitory substance was a fluorotricarboxylic acid. Subsequently it was shown that 2-fluorocitrate is indeed produced metabolically via the citrate synthase reaction (5) and that in the presence of this substance the enzyme aconitase is inhibited (6). Aconitase catalyzes the conversion of citrate to isocitrate (Iso) via the obligatory intermediate cis-aconitate (Scheme I).

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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 ...

During activation of aconitase a ferrous ion is incorporated into a [3Fe-4S] cluster to yield a structure with a [4Fe-4S] core. Using 57Fe or '"Fe for activation we have studied withMossbauer spectroscopy the beef heart enzyme in the presence of citrate. Our studies show that the environment of one iron site (Fea) of the [4Fe-4S] cluster is drastically altered in the presence of citrate. Fea is the iron acquired during activation of aconitase. (2) showed that it also contained labile sulfide but the potential implications of this discovery remained unknown and unexplored. Ruzicka and Beinert (3) then established a relationship between the oxidation state of the iron-sulfur (Fe-S) cluster and enzymatic activity, which seemed to suggest a regulatory function for the cluster in this enzyme. However, there remained uncertainties about the nature of the Fe-S cluster of aconitase (3)(4)(5) M6ssbauer spectroscopy requires the isotope 57Fe, which occurs at only 2.2% natural abundance. By using 57Fe enriched to >90% for the activation of aconitase we can follow specifically the newly incorporated Fe. By using the non-M6ssbauer isotope 56Fe for activation we can study the spectra of the other three sites, albeit with a much poorer signal-to-noise ratio. By this approach we have shown that doublet a represents the Fe acquired during activation (8). This Fe, designated here as Fea, is particularly labile and is lost, concomitant with enzyme activity, on oxidation by, e.g., oxygen or ferricyanide (10). Chemical analyses for Fe and labile S2-on the 3Fe and 4Fe enzymes together with results of extended x-ray absorption fine structure spectroscopy have provided strong evidence that the cluster remaining after loss of the labile Fe does not have the [3Fe-3S] structure, as originally assumed (7, 8), but retains its S2-in a [3Fe-4S] structure (11).The diamagnetic [4Fe-4S]2+ cluster of active aconitase can be reduced by one electron to the [4Fe-4S]+ state, which shows the typical g 1.94 (S = 1/2) EPR signal (8,12 Inasmuch as we see in these observations potential evidence for involvement of the Fe-S cluster in the reaction catalyzed by aconitase, we report here the relevant experiments and discuss their implications. 4674The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. § 1734 solely to indicate this fact.

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.(ABSTRACT TRUNCATED AT 250 WORDS)