Succinate-ubiquinone oxidoreductase (SQR) as part of the trichloroacetic acid cycle and menaquinol-fumarate oxidoreductase (QFR) used for anaerobic respiration by Escherichia coli are structurally and functionally related membrane-bound enzyme complexes. Each enzyme complex is composed of four distinct subunits. The recent solution of the X-ray structure of QFR has provided new insights into the function of these enzymes. Both enzyme complexes contain a catalytic domain composed of a subunit with a covalently bound flavin cofactor, the dicarboxylate binding site, and an iron-sulfur subunit which contains three distinct iron-sulfur clusters. The catalytic domain is bound to the cytoplasmic membrane by two hydrophobic membrane anchor subunits that also form the site(s) for interaction with quinones. The membrane domain of E. coli SQR is also the site where the heme b556 is located. The structure and function of SQR and QFR are briefly summarized in this communication and the similarities and differences in the membrane domain of the two enzymes are discussed.

Background Experimental and clinical evidence has pinpointed a critical role for matrix metalloproteinase-2 (MMP-2) in ischemic ventricular remodeling and systolic heart failure. Prior studies have demonstrated that transgenic expression of the full-length, 68 kDa, secreted form of MMP-2 induces severe systolic failure. These mice also had unexpected and severe mitochondrial structural abnormalities and dysfunction. We hypothesized that an additional intracellular isoform of MMP-2, which affects mitochondrial function is induced under conditions of systolic failure-associated oxidative stress. Methodology and Principal Findings Western blots of cardiac mitochondria from the full length MMP-2 transgenics, ageing mice and a model of accelerated atherogenesis revealed a smaller 65 kDa MMP-2 isoform. Cultured cardiomyoblasts subjected to transient oxidative stress generated the 65 kDa MMP-2 isoform. The 65 kDa MMP-2 isoform was also induced by hypoxic culture of cardiomyoblasts. Genomic database analysis of the MMP-2 gene mapped transcriptional start sites and RNA transcripts induced by hypoxia or epigenetic modifiers within the first intron of the MMP-2 gene. Translation of these transcripts yields a 65 kDa N-terminal truncated isoform beginning at M 77 , thereby deleting the signal sequence and inhibitory prodomain. Cellular trafficking studies demonstrated that the 65 kDa MMP-2 isoform is not secreted and is present in cytosolic and mitochondrial fractions, while the full length 68 kDa isoform was found only in the extracellular space. Expression of the 65 kDa MMP-2 isoform induced mitochondrial-nuclear stress signaling with activation of the pro-inflammatory NF-κB, NFAT and IRF transcriptional pathways. By microarray, the 65 kDa MMP-2 induces an innate immunity transcriptome, including viral stress response genes, innate immunity transcription factor IRF7, chemokines and pro-apoptosis genes. Conclusion A novel N-terminal truncated intracellular isoform of MMP-2 is induced by oxidative stress. This isoform initiates a primary innate immune response that may contribute to progressive cardiac dysfunction in the setting of ischemia and systolic failure.

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.

Factors governing the efficacy of long-range electron relays in enzymes have been examined using protein film voltammetry in conjunction with site-directed mutagenesis. Investigations of the fumarate reductase from Escherichia coli, in which three Fe-S clusters relay electrons over more than 30 A, lead to the conclusion that varying the medial [4Fe-4S] cluster potential over a 100 mV range does not have a significant effect on the inherent kinetics of electron transfer to and from the active-site flavin. The results support a proposal that the reduction potential of an individual electron relay site in a multicentered enzyme is not a strong determinant of activity; instead, as deduced from the potential dependence of catalytic electron transfer, electron flow through the intramolecular relay is rapid and reversible, and even uphill steps do not limit the catalytic rate.

We have examined the role of the quinone-binding (Q P ) site of Escherichia coli succinate:ubiquinone oxidoreductase (succinate dehydrogenase) in heme reduction and reoxidation during enzyme turnover. The SdhCDAB electron transfer pathway leads from a cytosolically localized flavin adenine dinucleotide cofactor to a Q P site located within the membrane-intrinsic domain of the enzyme. The Q P site is sandwiched between the [3Fe-4S] cluster of the SdhB subunit and the heme b 556 that is coordinated by His residues from the SdhC and SdhD subunits. The intercenter distances between the cluster, heme, and Q P site are all within the theoretical 14 Å limit proposed for kinetically competent intercenter electron transfer. Using EPR spectroscopy, we have demonstrated that the Q P site of SdhCDAB stabilized a ubisemiquinone radical intermediate during enzyme turnover. Potentiometric titrations indicate that this species has an E m,8 of ϳ60 mV and a stability constant (K STAB ) of ϳ1.0. Mutants of the following conserved Q P site residues, SdhC-S27, SdhC-R31, and SdhD-D82, have severe consequences on enzyme function. Mutation of the conserved SdhD-Y83 suggested to hydrogen bond to the ubiquinone cofactor had a less severe but still significant effect on function. In addition to loss of overall catalysis, these mutants also affect the rate of succinate-dependent heme reduction, indicating that the Q P site is an essential stepping stone on the electron transfer pathway from the [3Fe-4S] cluster to the heme. Furthermore, the mutations result in the elimination of EPR-visible ubisemiquinone during potentiometric titrations. Overall, these results demonstrate the importance of a functional, semiquinone-stabilizing Q P site for the observation of rapid succinate-dependent heme reduction.Escherichia coli encodes a tetrameric enzyme, succinate dehydrogenase (succinate:ubiquinone oxidoreductase; SQR) 2 , that is responsible for the oxidation of succinate to fumarate during aerobic growth. As a constituent of the bacterial inner membrane, SQR also interacts with the membrane-intrinsic quinone pool and couples the essential tricarboxylic acid cycle step of succinate oxidation to the respiratory electron transport chain (1). The structure of E. coli SQR has been solved by x-ray crystallography (2), and it displays remarkable structural and inferred functional similarity to the structures of the chicken (3) and pig (4) enzymes. As a model system for mitochondrial Complex II, E. coli SQR has the advantages of facile genetic manipulation, conservation of critical amino acid residues, and high level protein overexpression (5).E. coli SQR comprises four subunits, two of which, SdhA and SdhB, are hydrophilic and attached to the inner (cytoplasmic) surface of the plasma membrane via interactions with two hydrophobic membrane-intrinsic subunits, SdhC and SdhD (6). SdhA contains a redox active flavin adenine dinucleotide (FAD) at its dicarboxylic acid binding site that cycles via an EPR-visible flavin semiquinone between the FAD and FADH 2 r...

Mammalian complex I (NADH:ubiquinone oxidoreductase) exists as a mixture of interconvertible active (A) and de-activated (D) forms. The A-form is capable of NADH:quinone-reductase catalysis, but not the D-form. Complex I from the bacterium Paracoccus denitrificans, by contrast, exists only in the A-form. This bacterial complex contains 32 fewer subunits than the mammalian complex. The question arises therefore if the structural complexity of complex I from higher organisms correlates with its ability to undergo the A/D transition. In the present study, it was found that complex I from the bacterium Escherichia coli and from non-vertebrate organisms (earthworm, lobster, and cricket) did not show the A/D transitions. Vertebrate organisms (carp, frog, chicken), however, underwent similar A/D transitions to those of the well-characterized bovine complex I. Further studies showed that complex I from the lower eukaryotes, Neurospora crassa and Yarrowia lipolytica, exhibited very distinct A/D transitions with much lower activation barriers compared to the bovine enzyme. The A/D transitions of complex I as they relate to structure and regulation of enzymatic activity are discussed.

Succinate:quinone oxidoreductase (SQR, 4 succinate dehydrogenase) and menaquinol:fumarate oxidoreductase (QFR, fumarate reductase), members of the Complex II family, are homologous integral membrane proteins which couple the interconversion of succinate and fumarate with quinone and quinol (1-4). SQR is a key enzyme in the Krebs cycle, oxidizing succinate to fumarate during aerobic growth and reducing quinone to quinol and, thus, acts as a direct link between the Krebs cycle and the respiratory chain. QFR is found in anaerobic or facultative bacteria and lower eukaryotes, where it couples the oxidation of reduced quinones to the reduction of fumarate (1, 4). Escherichia coli SQR has four subunits, two hydrophilic subunits exposed to the cytoplasm (SdhA and SdhB), which interact with two hydrophobic membrane-intrinsic subunits (SdhC and SdhD) (5). SdhA contains the dicarboxylate-binding site and a covalently bound FAD cofactor which cycles between FAD and FADH 2 redox states during succinate oxidation (6). The electrons from succinate oxidation are sequentially transferred via a [2Fe-2S], a [4Fe-4S], and a [3Fe-4S] iron-sulfur cluster relay system in SdhB to a quinone-binding site (Q P ) located at the interface of the SdhB, SdhC, and SdhD subunits. SdhC and SdhD are both composed of three transmembrane helices and coordinate a low spin b-type heme via His residues contributed by each subunit (7,8).The first structural information about members of the Complex II family came from x-ray structures of the QFR enzymes from E. coli at 3.3 Å resolution (9) and Wolinella succinogenes at 2.2 Å resolution (10). These structures revealed details of the overall architecture of the subunits, the position of key redox cofactors, the electron transfer pathway, and the quinone-binding sites. At around the same time, the structures of soluble fumarate reductases found in anaerobic and microaerophilic bacteria and structurally homologous to the flavoprotein subunit of Complex II were solved by x-ray crystallography (1). Analysis of these soluble fumarate reductases has proven particularly informative in describing the mechanism of fumarate reduction and succinate oxidation at the dicarboxylate-binding site (11)(12)(13)(14).Structures of SQRs lagged behind those of the QFRs until the structure of the E. coli enzyme was solved at 2.6 Å (15). This structure, solved in space group R32, revealed that the E. coli enzyme is packed as a trimer. The structures of the SdhA and SdhB subunits were highly similar to those of E. coli and W. succinogenes QFRs, but the transmembrane SdhC and SdhD subunits showed differences compared with their QFR counterparts. The structure revealed the position of the redox sites and the dicarboxylate-and quinone-binding (Q) sites. The heme b molecule was shown to lie away from the electron transfer pathway, suggesting electrons are preferentially transferred from * This work was supported, in whole or in part, by National Institutes of Health Grant GM61606. This work was also supported by the Department of Ve...

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