Electron transfer in biological systems: an overview.
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Copper↗
Biomedical subjects
J L Dreyer
Publications and source records attributed to J L Dreyer.
The role of iron in the activation-inactivation of aconitase.
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.
Aconitate Hydratase↗
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.
Aconitate Hydratase↗
Iron-sulfur stoichiometry and structure of iron-sulfur clusters in three-iron proteins: evidence for [3Fe-4S] clusters.
Beef heart aconitase contains 3Fe clusters in its inactive and 4Fe clusters in its active form. The fully active form can be restored from the inactive one by insertion of Fe(2+), whereas S(2-) is not required. Chemical analyses for iron and labile sulfide yield Fe/S(2-) ratios of 0.66-0.74 for the inactive and 0.90-1.03 for the active form. Sulfane sulfur (S(0)) was not detected. We propose on the basis of these data that the inactive form may arise from the active one by loss of one iron only per cluster with the sulfur remaining as S(2-) in a [3Fe-4S] structure. Measurements by extended x-ray absorption fine structure (EXAFS) spectroscopy on the 3Fe form of aconitase yield a Fe..S distance of 2.24 A and a Fe..Fe distance of 2.71 A. This Fe..Fe distance is in agreement with that obtained by EXAFS on ferredoxin II of Desulfovibrio gigas, another 3Fe protein, but disagrees with Fe..Fe distances observed for the 3Fe cluster of Azotobacter vinelandii ferredoxin I by x-ray diffraction-namely, 4.1 A. We suggest that this difference may be due to the presence of a [3Fe-3S] structure in the Azotobacter ferredoxin I crystals vs. a [3Fe-4S] structure in liquid or frozen solutions of aconitase. The [3Fe-3S] cluster has been shown to have a relatively flat twist-boat structure, whereas a [3Fe-4S] cluster could be expected to essentially maintain the compact structure of the [4Fe-4S] cluster. This would explain the differences in Fe..Fe distances. Two possible structural models for a [3Fe-4S] cluster are discussed.
Aconitate Hydratase↗
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 = 2); the Mössbauer spectra exhibit al 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 either 27 Fe or 56 Fe 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.
Aconitate Hydratase↗
Relationship of the oxidation state of the iron-sulfur cluster of aconitase to activity and substrate binding.
It is known that aconitase from mammalian mitochondria is only partially active as isolated but may be activated by incubation with iron, ascorbate, and a thiol, or with dithionite. It has been suggested that the added Fe in the activation mixture is essential for activation and that it is incorporated in the enzyme [Villafranca, J. J., & Mildvan, A. S. (1971) J. Biol. Chem. 246, 772-779; Gawron, O., Waheed, A., Glaid, A. J., & Jaklitsch, A. (1974) Biochem. J. 139, 709-714]. However, it is shown in this paper that, when the enzyme has a full complement of 3Fe and 3S, full activation is reached coulometrically, without iron or other chemical reducing agents. It is clear, therefore, that the role of activators is to reduce the iron--sulfur cluster of the enzyme. The appearance of catalytic activity on reduction of the cluster shows a pronounced lag, as does the decay of activity after reoxidizing the cluster. This suggests that catalytic activity requires a conformational change in the protein which is initiated by reduction of the cluster and that, following reoxidation, activity disappears only after the inactive conformation is assumed. Citrate and the competitive inhibitor trans-aconitate are bound to a comparable extent to the active and inactive forms, but only the active form can bind 1-hydroxy-2-nitro-1,3-propanedicarboxylic acid, a transition-state analogue. This is interpreted to show that in the inactive state aconitase cannot enter the conformation it assumes in the transition state during catalysis.
Aconitate Hydratase↗
A rapid and simple method for the isolation of pure eosinophilic leukocytes from horse blood.
An improved and short method is described for the isolation of intact eosinophilic leukocytes from horse blood with high yield (1--1.5 g/20 l). Viability and purity of the preparations were verified by light and electron microscopy and by the trypan blue exclusion test. Isolated eosinophils were 98--100% pure, intact and viable, and they could be shown to phagocytise immune-complexes.
Animals↗