Background: Complex II is not considered a significant contributor to mitochondrial ROS production. Results: Complex II generates ROS in both the forward reaction, from succinate, and the reverse reaction, from the reduced ubiquinone pool. Conclusion: Occupancy and reduction state of the flavin dictate its ROS producing behavior. Significance: Based on the maximum rates observed, complex II may be a contributor to physiological ROS production.

In mitochondria, electrons derived from the oxidation of succinate by the tricarboxylic acid cycle enzyme succinate-ubiquinone oxido-reductase are transferred directly to the quinone pool. Here we provide evidence that the soluble form of this enzyme (succinate dehydrogenase) behaves as a diode that essentially allows electron flow in one direction only. The gating effect is observed when electrons are exchanged rapidly and directly between fully active succinate dehydrogenase and a graphite electrode. Turnover is therefore measured under conditions of continuously variable electrochemical potential. The otherwise rapid and efficient reduction of fumarate (the reverse reaction) is severely retarded as the driving force (overpotential) is increased. Such behaviour can arise if a rate-limiting chemical step like substrate binding or product release depends on the oxidation state of a redox group on the enzyme. The observation provides, for a biological electron-transport system, a simple demonstration of directionality that is enforced by kinetics as opposed to that which is assumed from thermodynamics.

Protein film voltammetry is used to probe the energetics of electron transfer and substrate binding at the active site of a respiratory flavoenzyme--the membrane-extrinsic catalytic domain of Escherichia coli fumarate reductase (FrdAB). The activity as a function of the electrochemical driving force is revealed in catalytic voltammograms, the shapes of which are interpreted using a Michaelis-Menten model that incorporates the potential dimension. Voltammetric experiments carried out at room temperature under turnover conditions reveal the reduction potentials of the FAD, the stability of the semiquinone, relevant protonation states, and pH-dependent succinate--enzyme binding constants for all three redox states of the FAD. Fast-scan experiments in the presence of substrate confirm the value of the two-electron reduction potential of the FAD and show that product release is not rate limiting. The sequence of binding and protonation events over the whole catalytic cycle is deduced. Importantly, comparisons are made with the electrocatalytic properties of SDH, the membrane-extrinsic catalytic domain of mitochondrial complex II.

Site-directed mutants of Escherichia coli fumarate reductase in which each of the four N-terminal cysteine residues in the FrdB subunit, residues 57, 62, 65, and 77, was mutated individually to serine have been constructed, overexpressed, and investigated in terms of enzymatic activity as well as the EPR and redox properties of the iron-sulfur centers. In each case, the mutant contains a functional fumarate reductase in which all three of the constituent iron-sulfur clusters (i.e., The menaquinol-fumarate oxidoreductase (EC 1.3.99.1) of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor (1, 2). The membrane-extrinsic fumarate reductase domain comprises a flavoprotein (Fp), FrdA (66 kDa), with a covalently bound FAD (3), and an iron-sulfur protein (Ip), FrdB (27 kDa). Two small hydrophobic peptides (4), FrdC (15 kDa) and FrdD (13 kDa), anchor the enzyme to the membrane and are essential for interaction with quinones (5-7). The combination ofmagnetic CD and EPR spectroscopies has provided evidence for three types of iron-sulfur clusters, each stoichiometric with FAD, in the two-subunit fumarate reductase: center 1, [2Fe-

The posttranscriptional control of iron uptake, storage, and utilization by iron-responsive elements (IREs) and iron regulatory proteins (IRPs) provides a molecular framework for the regulation of iron homeostasis in many animals. We have identified and characterized IREs in the mRNAs for two different mitochondrial citric acid cycle enzymes. Drosophila melanogaster IRP binds to an IRE in the 5' untranslated region of the mRNA encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH). This interaction is developmentally regulated during Drosophila embryogenesis. In a cell-free translation system, recombinant IRP-1 imposes highly specific translational repression on a reporter mRNA bearing the SDH IRE, and the translation of SDH-Ip mRNA is iron regulated in D. melanogaster Schneider cells. In mammals, an IRE was identified in the 5' untranslated regions of mitochondrial aconitase mRNAs from two species. Recombinant IRP-1 represses aconitase synthesis with similar efficiency as ferritin IRE-controlled translation. The interaction between mammalian IRPs and the aconitase IRE is regulated by iron, nitric oxide, and oxidative stress (H202), indicating that these three signals can control the expression of mitochondrial aconitase mRNA. Our results identify a regulatory link between energy and iron metabolism in vertebrates and invertebrates, and suggest biological functions for the IRE/IRP regulatory system in addition to the maintenance of iron homeostasis.Most animals, including humans and other mammals, frogs, fish, and flies appear to regulate cellular iron metabolism posttranscriptionally by means of the interaction of ironresponsive elements (IREs) and iron regulatory proteins (IRPs) (1, 2). In mammals, where this system is best characterized, IREs regulate the mRNAs for the iron storage protein ferritin, the receptor for cellular iron uptake (transferrin receptor) and the rate limiting enzyme for the main iron utilization pathway, erythroid 5-aminolevulinate synthase (eALAS). By binding to IREs in the 5' untranslated regions (UTRs) of ferritin and eALAS mRNAs, IRP-1 and, independently, IRP-2 (3, 4) repress the translation of these transcripts (5-9), whereas the transferrin receptor mRNA is stabilized by IRP binding to IREs located in the 3' UTR (10-12). IRP-1 and IRP-2 bind to IREs in iron deficient, but not in iron replete, cells (3,13,14). IRE/IRP interactions thus serve to maintain iron homeostasis by coordinated regulation of iron uptake, storage, and utilization. Moreover, the regulation of IRP-1 by nitric oxide (NO) and H202 and of IRP-2 by [15][16][17][18][19] connects the regulation of iron metabolism to additional signaling pathways (K.P., G. Weiss, and M.W.H., unpublished work).In addition to the mRNAs encoding proteins central to iron metabolism, an IRE motif was identified in the 5' UTR of porcine mitochondrial aconitase mRNA (20). Like the other citric acid cycle enzymes, aconitase is nuclear-encoded and translated in the cytoplasm. Following their posttranslational imp...

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Complex II (succinate:quinone oxidoreductase) of aerobic respiratory chains oxidizes succinate to fumarate and passes the electrons directly into the quinone pool. It serves as the only direct link between activity in the citric acid cycle and electron transport in the membrane. Finer details of these reactions and interactions are but poorly understood. However, complex II has extremely similar structural and catalytic properties to quinol :fumarate oxidoreductases of anaerobic organisms, for which X-ray structures have recently become available. These offer new insights into structure^function relationships of this class of flavoenzymes, including evidence favoring protein movement during catalysis.z 2000 Federation of European Biochemical Societies.

The succinate dehydrogenases (SDH: soluble, membrane-extrinsic subunits of succinate:quinone oxidoreductases) from Escherichia coli and beef heart mitochondria each adsorb at a pyrolytic graphite 'edge' electrode and catalyse the interconversion of succinate and fumarate according to the electrochemical potential that is applied. E. coli and beef heart mitochondrial SDH share only ca. 50% homology, yet the steady-state catalytic activities, when measured over a continuous potential range, display very similar catalytic operating potentials and energetic biases (the relative ability to catalyse succinate oxidation vs. fumarate reduction). Importantly, E. coli SDH also exhibits the interesting 'tunnel-diode' behaviour previously reported for the mitochondrial enzyme. Thus as the potential is lowered below ca. -60 mV (pH 7, 38 degrees C) the rate of catalytic fumarate reduction decreases abruptly despite an increase in driving force. Since the homology relates primarily to residues associated with active site regions, the marked similarity in the voltammetry reaffirms our previous conclusions that the tunnel-diode behaviour is a characteristic property of the enzyme active site. Thus, succinate dehydrogenase is an excellent fumarate reductase, but its activity in this direction is limited to a very specific range of potential.