Light-harvesting complex II binds to several small subunits of photosystem I

Mobile light-harvesting complex II (LHCII) is implicated in the regulation of excitation energy distribution between Photosystem I (PSI) and Photosystem II (PSII) during state transitions. To investigate how LHCII interacts with PSI during state transitions, PSI was isolated from Arabidopsis thaliana plants treated with PSII or PSI light. The PSI preparations were made using digitonin. Chemical cross-linking using dithio-bis(succinimidylpropionate) followed by diagonal electrophoresis and immunoblotting showed that the docking site of LHCII (Lhcb1) on PSI is comprised of the PSI-H, -L, and -I subunits. This was confirmed by the lack of energy transfer from LHCII to PSI in the digitonin-PSI isolated from plants lacking PSI-H and -L. Digitonin-PSI was purified further to obtain an LHCII.PSI complex, and two to three times more LHCII was associated with PSI in the wild type in State 2 than in State 1. Lhcb1 was also associated with PSI from plants lacking PSI-K, but PSI from PSI-H, -L, or -O mutants contained only about 30% of Lhcb1 compared with the wild type. Surprisingly, a significant fraction of the LHCII bound to PSI in State 2 was not phosphorylated. Cross-linking prior to sucrose gradient purification resulted in copurification of phosphorylated LHCII in the wild type, but not with PSI from the PSI-H, -L, and -O mutants. The data suggest that migration of LHCII during state transitions cannot be explained sufficiently by different affinity of phosphorylated and unphosphorylated LHCII for PSI but is likely to involve structural changes in thylakoid organization.     

Generation of superoxide by the mitochondrial Complex I

Superoxide production by inside-out coupled bovine heart submitochondrial particles, respiring with succinate or NADH, was measured. The succinate-supported production was inhibited by rotenone and uncouplers, showing that most part of superoxide produced during succinate oxidation is originated from univalent oxygen reduction by Complex I. The rate of the superoxide (O2*-)) production during respiration at a high concentration of NADH (1 mM) was significantly lower than that with succinate. Moreover, the succinate-supported O2*- production was significantly decreased in the presence of 1 mM NADH. The titration curves, i.e., initial rates of superoxide production versus NADH concentration, were bell-shaped with the maximal rate (at 50 microM NADH) approaching that seen with succinate. Both NAD+ and acetyl-NAD+ inhibited the succinate-supported reaction with apparent Ki's close to their Km's in the Complex I-catalyzed succinate-dependent energy-linked NAD+ reduction (reverse electron transfer) and NADH:acetyl-NAD+ transhydrogenase reaction, respectively. We conclude that: (i) under the artificial experimental conditions the major part of superoxide produced by the respiratory chain is formed by some redox component of Complex I (most likely FMN in its reduced or free radical form); (ii) two different binding sites for NADH (F-site) and NAD+ (R-site) in Complex I provide accessibility of the substrates-nucleotides to the enzyme red-ox component(s); F-site operates as an entry for NADH oxidation, whereas R-site operates in the reverse electron transfer and univalent oxygen reduction; (iii) it is unlikely that under the physiological conditions (high concentrations of NADH and NAD+) Complex I is responsible for the mitochondrial superoxide generation. We propose that the specific NAD(P)H:oxygen superoxide (hydrogen peroxide) producing oxidoreductase(s) poised in equilibrium with NAD(P)H/NAD(P)+ couple should exist in the mitochondrial matrix, if mitochondria are, indeed, participate in ROS-controlled processes under physiologically relevant conditions.     

Functional specialization of maize mitochondrial aldehyde dehydrogenases

The maize (Zea mays) rf2a and rf2b genes both encode homotetrameric aldehyde dehydrogenases (ALDHs). The RF2A protein was shown previously to accumulate in the mitochondria. In vitro import experiments and ALDH assays on mitochondrial extracts from rf2a mutant plants established that the RF2B protein also accumulates in the mitochondria. RNA gel-blot analyses and immunohistolocation experiments revealed that these two proteins have only partially redundant expression patterns in organs and cell types. For example, RF2A, but not RF2B, accumulates to high levels in the tapetal cells of anthers. Kinetic analyses established that RF2A and RF2B have quite different substrate specificities; although RF2A can oxidize a broad range of aldehydes, including aliphatic aldehydes and aromatic aldehydes, RF2B can oxidize only short-chain aliphatic aldehydes. These two enzymes also have different pH optima and responses to changes in substrate concentration. In addition, RF2A, but not RF2B or any other natural ALDHs, exhibits positive cooperativity. These functional specializations may explain why many species have two mitochondrial ALDHs. This study provides data that serve as a basis for identifying the physiological pathway by which the rf2a gene participates in normal anther development and the restoration of Texas cytoplasm-based male sterility. For example, the observations that Texas cytoplasm anthers do not accumulate elevated levels of reactive oxygen species or lipid peroxidation and the kinetic features of RF2A make it unlikely that rf2a restores fertility by preventing premature programmed cell death.     

Regulation of mitochondrial dehydrogenases by calcium ions

Studies in Bristol in the 1960s and 1970s, led to the recognition that four mitochondrial dehydrogenases are activated by calcium ions. These are FAD-glycerol phosphate dehydrogenase, pyruvate dehydrogenase, NAD-isocitrate dehydrogenase and oxoglutarate dehydrogenase. FAD-glycerol phosphate dehydrogenase is located on the outer surface of the inner mitochondrial membrane and is influenced by changes in cytoplasmic calcium ion concentration. The other three enzymes are located within mitochondria and are regulated by changes in mitochondrial matrix calcium ion concentration. These and subsequent studies on purified enzymes, mitochondria and intact cell preparations have led to the widely accepted view that the activation of these enzymes is important in the stimulation of the respiratory chain and hence ATP supply under conditions of increased ATP demand in many stimulated mammalian cells. The effects of calcium ions on FAD-isocitrate dehydrogenase involve binding to an EF-hand binding motif within this enzyme but the binding sites involved in the effects of calcium ions on the three intramitochondrial dehydrogenases remain to be fully established. It is also emphasised in this article that these three dehydrogenases appear only to be regulated by calcium ions in vertebrates and that this raises some interesting and potentially important developmental issues.     

Aflatoxin interactions with rat hepatic mitochondrial dehydrogenases

The effects of sublethal and possibly carcinogenic concentrations of aflatoxins B₁, B₂, G₁, G₂, and M₁ (AFB₁, AFG₂, AFG₁, AFG₂, and AFM₁) on rat hepatic mitochondrial dehydrogenases were studied by redox-dye titration. All the aflatoxins (minus AFG₂) enhanced the activities of the matrix dehydrogenases but had no marked effects on the membrane-bound enzymes. However, mitochondrial preincubation with each toxin raised the activities of all the dehydrogenases. AFG₂ was unique by inhibiting all the enzymes irreversibly.     

Complex I dysfunction redirects cellular and mitochondrial metabolism in Arabidopsis

Mitochondrial complex I is a major avenue for reduced NAD oxidation linked to oxidative phosphorylation in plants. However, the plant enzyme has structural and functional features that set it apart from its counterparts in other organisms, raising questions about the physiological significance of this complex in plants. We have developed an experimental model in which rotenone, a classic complex I inhibitor, has been applied to Arabidopsis (Arabidopsis thaliana) cell suspension cultures in order to dissect early metabolic adjustments involved in cell acclimation to mitochondrial dysfunction. Rotenone induced a transitory decrease in cellular respiration (0-4 h after treatment). Cell respiration then progressively recovered and reached a steady state at 10 to 12 h after treatment. Complex I inhibition by rotenone did not induce obvious oxidative stress or cell death but affected longer term cell growth. Integrated analyses of gene expression, the mitochondrial proteome, and changes in primary metabolism indicated that rotenone treatment caused changes in mitochondrial function via alterations in specific components. A physical disengagement of glycolytic activities associated with the mitochondrial outer membrane was observed, and the tricarboxylic acid cycle was altered. Amino acid and organic acid pools were also modified by rotenone treatment, with a marked early decrease of 2-oxoglutarate, aspartate, and glutamine pools. These data demonstrate that, in Arabidopsis cells, complex I inhibition by rotenone induces significant remodeling of metabolic pathways involving the mitochondria and other compartments and point to early metabolic changes in response to mitochondrial dysfunction.     

Catalytic properties of mitochondrial NADH-ubiquinone reductase (Complex I)

Qualitative and quantitative characteristics of the reactions catalyzed by the most complex and least understood proton translocating unit of the mammalian respiratory chain (NADH-ubiquinone oxidoreductase, Complex I) are described for enzyme preparations differing in degree of resolution--from intact mitochondria to homogeneous small enzyme fragments. Special attention is given to the problems and pitfalls of reliable interpretation of the kinetic analysis of the enzyme activities. Detailed analysis of the problems concerning the slow active/inactive reversible enzyme transition is provided.     

Immunological analysis of plant mitochondrial NADH dehydrogenases.

Plant mitochondrial NADH dehydrogenases were analysed by two immunological strategies. The first exploited an antiserum raised to a preparation of SDS-solubilized mitochondrial-inner-membrane particles. By using a combination of activity-immunoprecipitation and crossed immunoelectrophoresis, it was shown that Triton X-100-solubilized membranes contain at least three immunologically distinct NADH dehydrogenases. Two of these were subsequently isolated by line immunoelectrophoresis and analysed for polypeptide composition: one contained three polypeptides with molecular masses of 75, 62 and 41 kDa; the other was a single polypeptide with a molecular mass of 53 kDa. The other approach was to probe plant mitochondrial membranes with antibodies raised to a purified preparation of ox heart rotenone-sensitive NADH dehydrogenase and subunits thereof. Cross-reactions were observed with the subunit-specific antisera against the 30 and 49 kDa ox heart proteins. However, the molecular masses of the equivalent polypeptides in plant mitochondria are slightly lower, at 27 and 46 kDa respectively.     

Sodium dodecyl sulfate, concurrent inhibitor of several dehydrogenases

The effect of sodium dodecyl sulfate on the activity of highly purified or crystalline enzymes has been studied. The enzymes were: lactate dehydrogenase (LDH), malate dehydrogenase (MDH). isocitrate dehydrogenase (ICDH), glucose-6-phosphate dehydrogenase (G6P-DH), lipase, alkaline phosphatase. Sodium dodecyl sulfate, always under the critical micellar concentration, shows a selective inhibitory effect. A kinetic analysis of the inhibitory action on LDH, MDH, ICDH and G6P-DH was also carried out.     

An ubiquinone-binding protein in mitochondrial NADH-ubiquinone reductase (Complex I)

An ubiquinone-binding protein (QP) was purified from mitochondrial NADH-ubiquinone reductase (Complex I). Complex I was separated into 3 fragments: a fraction of hydrophobic proteins, that of soluble iron-sulfur protein (IP) and soluble NADH dehydrogenase of flavoprotein by a procedure involving the resolution with DOC and cholate, followed by ethanol and ammonium acetate fractionations. About 40% of the total ubiquinone was recovered in the IP fragment which consisted of 12 polypeptides. The QP was purified from the IP fragment with a hydrophobic affinity chromatography. SDS-polyacrylamide gel electrophoresis showed that the purified QP corresponded to 14-kDa polypeptide of the IP fragment and was a different protein from the QP (12.4 kDa) in Complex III. The purified QP (14 kDa) contained one mol ubiquinone per mol. The ubiquinone-depleted IP fragment could rebind ubiquinone. These results indicate that an ubiquinone-binding site in Complex I is on the 14-kDa polypeptide of the IP fragment.     

Redox cycling of adrenaline and adrenochrome catalysed by mitochondrial Complex I

Complex I in bovine heart submitochondrial particles catalyses the NADH-supported generation of superoxide anion; adrenaline is oxidised by superoxide to adrenochrome that, on its hand, is reduced by Complex I, thus establishing a redox cycle that amplifies the superoxide production. The routes in Complex I for superoxide formation and for adrenochrome reduction appear to be different, since they have a different sensitivity to Complex I inhibitors. The results are discussed in terms of current assays for superoxide detection and of pathologies linked to catecholamine oxidation.     

Transhydrogenation reactions catalyzed by mitochondrial NADH-ubiquinone oxidoreductase (Complex I)

NADH-ubiquinone oxidoreductase (complex I) is the first enzyme of the respiratory electron transport chain in mitochondria. It conserves the energy from NADH oxidation, coupled to ubiquinone reduction, as a proton motive force across the inner membrane. Complex I catalyzes NADPH oxidation, NAD+ reduction, and hydride transfers from reduced to oxidized nicotinamide nucleotides also. Here, we investigate the transhydrogenation reactions of complex I, using four different nucleotide pairs to encompass a range of reaction rates. Our experimental data are described accurately by a ping-pong mechanism with double substrate inhibition. Thus, we contend that complex I contains only one functional nucleotide binding site, in agreement with recent structural information, but in disagreement with previous mechanistic models which have suggested that two different binding sites are employed to catalyze the two half reactions. We apply the Michaelis-Menten equation to describe the productive states formed when the nucleotide and the active-site flavin mononucleotide have complementary oxidation states, and dissociation constants to describe the nonproductive states formed when they have the same oxidation state. Consequently, we derive kinetic and thermodynamic information about nucleotide binding and interconversion in complex I, relevant to understanding the mechanisms of coupled NADH oxidation and NAD+ reduction, and to understanding how superoxide formation by the reduced flavin is controlled. Finally, we discuss whether NADPH oxidation and/or transhydrogenation by complex I are physiologically relevant processes.     

Yeast mitochondrial outer membrane specifically binds cytoplasmically-synthesized precursors of mitochondrial proteins

The precursor of cytochrome b₂ (a cytoplasmically-synthesized mitochondrial protein) binds to isolated mitochondria or to isolated outer membrane vesicles. Binding does not require an energized inner membrane, is diminished by trypsin treatment of the membranes and is not observed with the partially processed (intermediate) form of the cytochrome b₂ precursor or with non-mitochondrial proteins. Upon energization of the mitochondria, the bound precursor is imported and cleaved to the mature form. Similar results were obtained with the precursor of citrate synthase. This receptor-like binding activity was present in isolated outer, but not inner membrane. It was solubilized from outer membrane with non-ionic detergent and reconstituted into liposomes.     

Yeast mitochondrial dehydrogenases are associated in a supramolecular complex

Separation of yeast mitochondrial complexes by colorless native polyacrylamide gel electrophoresis led to the identification of a supramolecular structure exhibiting NADH-dehydrogenase activity. Components of this complex were identified by N-terminal Edman degradation and matrix-assisted laser desorption ionization mass spectrometry. The complex was found to contain the five known intermembrane space-facing dehydrogenases, namely two external NADH-dehydrogenases Nde1p and Nde2p, glycerol-3-phosphate dehydrogenase Gut2p, D- and L-lactate-dehydrogenases Dld1p and Cyb2p, the matrix-facing NADH-dehydrogenase Ndi1p, two probable flavoproteins YOR356Wp and YPR004Cp, four tricarboxylic acids cycle enzymes (malate dehydrogenase Mdh1p, citrate synthase Cit1p, succinate dehydrogenase Sdh1p, and fumarate hydratase Fum1p), and the acetaldehyde dehydrogenase Ald4p. The association of these proteins is discussed in terms of NADH-channeling.