Variation in the organization and subunit composition of the mammalian pyruvate dehydrogenase complex E2/E3BP core assembly.

Crucial to glucose homoeostasis in humans, the hPDC (human pyruvate dehydrogenase complex) is a massive molecular machine comprising multiple copies of three distinct enzymes (E1-E3) and an accessory subunit, E3BP (E3-binding protein). Its icosahedral E2/E3BP 60-meric 'core' provides the central structural and mechanistic framework ensuring favourable E1 and E3 positioning and enzyme co-operativity. Current core models indicate either a 48E2+12E3BP or a 40E2+20E3BP subunit composition. In the present study, we demonstrate clear differences in subunit content and organization between the recombinant hPDC core (rhPDC; 40E2+20E3BP), generated under defined conditions where E3BP is produced in excess, and its native bovine (48E2+12E3BP) counterpart. The results of the present study provide a rational basis for resolving apparent differences between previous models, both obtained using rhE2/E3BP core assemblies where no account was taken of relative E2 and E3BP expression levels. Mathematical modelling predicts that an 'average' 48E2+12E3BP core arrangement allows maximum flexibility in assembly, while providing the appropriate balance of bound E1 and E3 enzymes for optimal catalytic efficiency and regulatory fine-tuning. We also show that the rhE2/E3BP and bovine E2/E3BP cores bind E3s with a 2:1 stoichiometry, and propose that mammalian PDC comprises a heterogeneous population of assemblies incorporating a network of E3 (and possibly E1) cross-bridges above the core surface.

Reconstitution of the mitochondrial PrxIII antioxidant defence pathway: general properties and factors affecting PrxIII activity and oligomeric state.

The mitochondrial 2-Cys peroxiredoxin PrxIII serves as a thioredoxin-dependent peroxidase operating in tandem with its cognate partners, an organelle-specific thioredoxin (Trx2) and NADP-linked thioredoxin reductase (TRR2). This PrxIII pathway is emerging as a primary regulator of intracellular H(2)O(2) levels with dual roles in antioxidant defence and H(2)O(2)-mediated signalling. Here we describe the reconstitution of the mammalian PrxIII pathway in vitro from its purified recombinant components and investigate some of its overall properties. Employing the site-directed PrxIII mutants C47S, C66S and C168S, the putative N and C-terminal catalytic cysteine residues are shown to be essential for function whereas the C66S mutant retains full activity. The pathway attains maximal capacity at low H(2)O(2) concentrations (<10 microM) and is progressively inhibited in the range 0.1 mM to 1.0 mM peroxide. Damage to PrxIII caused by over-oxidation is confirmed by the appearance of abnormal oxidised species of PrxIII on SDS-PAGE at elevated H(2)O(2) levels. The presence of an N-terminal His-tag on PrxIII markedly enhances dodecamer stability, particularly apparent in its oxidised state. Its removal promotes oxidised PrxIII dissociation into dimers and leads to a 3.0-3.5-fold stimulation in peroxidase activity. The unusual concatenated crystal structure of PrxIII consisting of two-interlocked dodecameric rings is also evident in dilute solution employing transmission electron microscopy; however, it represents only 3-5% of the population with most molecules present as single toroids. Moreover, concatenated PrxIII C168S reverts to single toroids on crystal dissolution indicating that these higher-order structures are produced dynamically during the crystallisation process.

Mitochondrial peroxiredoxins.

Mitochondria are the major intracellular sites of oxygen consumption producing reactive oxygen species (ROS) as toxic by-products of oxidative phosphorylation, primarily via electron leakage from the respiratory chain. The resultant types of chemical damage to lipids, DNA and proteins are described as well as the broader implications for the involvement of ROS in disease onset and progression. The relative contributions of mitochondrial, enzyme-linked, antioxidant defence systems to tissue protection are also reviewed as is the emerging importance of the peroxiredoxin family in general to H2O2-mediated signalling The constituent enzymes of the mitochondrial PrxIII pathway are discussed in detail including the roles of PrxIII and PrxV in their capacities as typical 2-cys and atypical 2-cys thioredoxin-dependent hydroperoxide reductases, respectively. The structures and catalytic mechanisms of PrxIII and V are examined and some key properties of the reconstituted mitochondrial PrxIII pathway are highlighted with specific reference to the susceptibility of peroxiredoxins to inactivation at elevated H2O2 levels and their potential for participation in H2O2-mediated signalling responses. It is concluded that mitochondrial Prxs form a vital link in an integrated cellular antioxidant defence network that minimises ROS-mediated damage and ensures that cells mount appropriate responses to increased levels of oxidative stress via the upregulation of key cell signalling pathways.

Bovine mitochondrial peroxiredoxin III forms a two-ring catenane.

A crystal structure is reported for the C168S mutant of a typical 2-Cys peroxiredoxin III (Prx III) from bovine mitochondria at a resolution of 3.3 A. Prx III is present as a two-ring catenane comprising two interlocking dodecameric toroids that are assembled from basic dimeric units. Each ring has an external diameter of 150 A and encompasses a central cavity that is 70 A in width. The concatenated dodecamers are inclined at an angle of 55 degrees, which provides a large contact surface between the rings. Dimer-dimer contacts involved in toroid formation are hydrophobic in nature, whereas the 12 areas of contact between interlocked rings arise from polar interactions. These two major modes of subunit interaction provide important insights into possible mechanisms of catenane formation.

Improved Catenated Structures of Bovine Peroxiredoxin III F190L Reveal Details of Ring-Ring Interactions and a Novel Conformational State.

Mitochondrial 2-cys peroxiredoxin III (PrxIII) is a key player in antioxidant defence reducing locally-generated H2O2 to H2O. A Phe to Leu (F190L) mutation in the C-terminal α-helix of PrxIII, mimicking that found in some bacteria and parasites, increases its resistance to hyperoxidation but has no obvious influence on peroxidase activity. Here we report on the oxidized and reduced crystal structures of bovine PrxIII F190L at 2.4 Å and 2.2 Å, respectively. Both structures exist as two-ring catenanes with their dodecameric rings inclined at 55o to each other, similar to that previously reported for PrxIII C168S. The new higher-resolution structures reveal details of the complex network of H-bonds stabilising the inter-toroid contacts. In addition, Arg123, the key conserved residue, that normally interacts with the catalytic cys (Cp, cys 47) is found in a distinct conformation extending away from the Cp while the characteristic Arg-Glu-Arg network, underpinning the active-site geometry also displays a distinctive arrangement, not observed previously. This novel active-site organisation may provide new insights into the dynamics of the large-scale conformational changes occurring between oxidized and reduced states.

Age-related increase in 4-hydroxynonenal adduction to rat heart alpha-ketoglutarate dehydrogenase does not cause loss of its catalytic activity.

4-hydroxynonenal (HNE), a product of omega-6 polyunsaturated fatty acid peroxidation, impairs mitochondrial respiration in vitro by adducting the alpha-ketoglutarate dehydrogenase complex (KGDC) and inhibiting its activity. The present study seeks to define whether aging increases HNE adduction to rat heart KGDC, and whether such adduction impacts KGDC activity. We found that hearts from old rats exhibit significantly (p< or =0.01) higher HNE-modified mitochondrial proteins when compared with those from young rats. Among these proteins, dihydrolipoamide succinyltransferase, the E2k component of KGDC, was most markedly modified (p< or =0.01) by HNE with age. As opposed to that seen in vitro, no significant change in electrophoretic mobility or impairment in enzyme activity was observed. On the contrary, KGDC activity increased onefold (p< or =0.01) in old rats, suggesting that the aging myocardium is not affected by HNE adduction or compensates for such damage. Further analysis revealed that heightened KGDC activity was not due to increased protein content or gene expression, but correlates with a lower Km for alpha-ketoglutarate. Thus, contrary to that observed in vitro, the measurement of HNE-KGDC adduct in rat heart is more relevant as a marker of age-related protein oxidation than a factor of mitochondrial dysfunction.

Cellular mitochondrial heterogeneity in cultured astrocytes as demonstrated by immunogold labeling of alpha-ketoglutarate dehydrogenase.

In brain cells, various metabolites and metabolic pathways, largely of mitochondrial origin, have been shown to be compartmentalized. Attention has therefore been focused on the possible existence of mitochondrial heterogeneity in the brain at the cellular level. To determine whether mitochondria in cultured cortical and cerebellar astrocytes are heterogeneous at the single cell level, immunogold electron microscopy and an antibody against the alpha-ketoglutarate dehydrogenase component of the alpha-ketoglutarate dehydrogenase complex, a marker enzyme for the tricarboxylic acid (TCA) cycle, were employed. The number of gold particles was counted in the mitochondria of 36 and 42 cells from cultured cerebellar and cortical astrocytes, respectively. A test for random distribution (Poisson distribution) of mitochondria according to the number of gold particles was subsequently performed for every one of the 36 and 42 cells as the ratio variance/mean (= index of dispersion). This should be approximately distributed as chi2/degrees of freedom (df) = n - 1, n = number of mitochondria), if the observations obeyed a Poisson distribution. For 26 of the 36 (cerebellar astrocytes) distributions and for 28 of the 42 (cortical astrocytes) distributions a random distribution had to be rejected. These findings therefore strongly indicate that alpha-ketoglutarate dehydrogenase is heterogeneously distributed in mitochondria within individual astrocytes originating either from cerebellum or cerebral cortex. In conclusion, this study underlines the probability that mitochondrial heterogeneity at the single cell level might be extended to involve other metabolic pathways and metabolites.

Structure-function analysis of recombinant substrate protein 22 kDa (SP-22). A mitochondrial 2-CYS peroxiredoxin organized as a decameric toroid.

Bovine mitochondrial SP-22 is a member of the peroxiredoxin family of peroxidases. It belongs to the peroxiredoxin 2-Cys subgroup containing three cysteines at positions 47, 66, and 168. The cloning and overexpression in Escherichia coli of recombinant wild type SP-22 and its three cysteine mutants (C47S, C66S, and C168S) are reported. Purified His-tagged SP-22 was fully active with Cys-47 being confirmed as the catalytic residue. The enzyme forms a stable decameric toroid consisting of five basic dimeric units containing intermolecular disulfide bonds linking the catalytically active Cys-47 of one subunit and Cys-168 of the adjacent monomer. The disulfide bonds are not required for overall structural integrity. The toroidal units have average external and internal diameters of 15 and 7 nm, respectively, and can form stacks in a lateral arrangement of two or three rings. C47S had a pronounced tendency to stack in long tubular structures containing up to 60 rings. Further unusual structural features are the presence of radial spikes projecting from the external surface and ordered electron-dense material within the central cavity of the toroid.