BBA - Bioenergetics (v.1658, #3)

In a majority of living organisms, a fundamental protection mechanism from reactive oxygen species is by the ascorbate–glutathione cycle in which an important antioxidant, ascorbate (vitamin C), is utilized to convert harmful H2O2 to H2O. Monodehydroascorbate reductase (MDHAR) maintains reduced pools of ascorbate by recycling the oxidized form of ascorbate. By screening a Brassica campestris cDNA library, we identified a B. campestris MDHAR cDNA (BcMdhar) which encodes a polypeptide of 434 amino acids possessing domains characteristic of FAD- and NAD(P)H-binding proteins. The predicted amino acid sequence of the open reading frame (ORF) shows a high level of identity to the cytosolic MDHAR of rice, pea and tomato, and does not possess N-terminal leader sequence suggesting that it encodes a cytosolic form of MDHAR. Genomic Southern blot analysis indicated that a single nuclear gene encodes this enzyme. Northern hybridization analysis detected BcMdhar transcripts in all plant tissues examined. The level of BcMdhar mRNA increased in response to oxidative stress invoked by hydrogen peroxide, salicylic acid, paraquat, and ozone.
Keywords: Antioxidant; Ascorbate–glutathione cycle; Monodehydroascorbate reductase; Oxidative stress; Brassica campestris;

Mitochondrial alterations and apoptosis in smooth muscle from aged rats by Guiomar S. Lopes; Oswaldo A. Mora; Paulo Cerri; Flávio P. Faria; Neide H. Jurkiewicz; Aron Jurkiewicz; Soraya S. Smaili (187-194).
We studied changes in mitochondrial morphology and function in the smooth muscle of rat colon. Under confocal microscopy, tissues loaded with potentiometric dye displayed rapid and spontaneous depolarization. Cyclosporin A (CsA), inhibitor of the permeability transition pore (PTP), caused an increase in mitochondrial membrane potential (ΔΨ m) in tissues from adult young animals. In aged rats these changes were not observed. This suggests that physiological activation of PTP in aged rats is reduced. Electron microscopy showed alterations of the mitochondrial ultrastructure in tissues from aged rats involving a decreased definition of the cristae and fragmentation of the mitochondrial membranes. We also detected an increase in apoptotic cells in the smooth muscle from aged animals. Our results show that the aging process changes PTP activity, the ability to maintain ΔΨ m and mitochondrial morphology. It is suggested that these can be associated with mitochondrial damage and cell death.
Keywords: Mitochondria; Calcium; Smooth muscle; Aging; Mitochondrial dysfunction;

On the mechanism of mitochondrial permeability transition induction by glycyrrhetinic acid by Cristina Fiore; Mauro Salvi; Mario Palermo; Giulietta Sinigaglia; Decio Armanini; Antonio Toninello (195-201).
Glycyrrhetinic acid (GE), the aglycone of glycyrrhizic acid, a triterpene glycoside which represents one of the main constituents of licorice root, induces an oxidative stress in liver mitochondria responsible for the induction of membrane permeability transition. In fact, GE, by interacting with the mitochondrial respiratory chain, generates hydrogen peroxide which in turn oxidizes critical thiol groups and endogenous pyridine nucleotides leading to the opening of the transition pore. Most likely the reactive group of GE is the carbonyl oxygen in C-11 which, by interacting mainly with a Fe/S centre of mitochondrial complex I, generates an oxygen-centered radical responsible for the pro-oxidant action.
Keywords: Mitochondria; Glycyrrhetinic acid; Electron transport chain; Reactive oxygen species; Permeability transition;

Dimeric H+-ATP synthase in the chloroplast of Chlamydomonas reinhardtii by Sascha Rexroth; Jürgen M.W. Meyer zu Tittingdorf; Helena J. Schwaßmann; Frank Krause; Holger Seelert; Norbert A. Dencher (202-211).
H+-ATP synthase is the dominant ATP production site in mitochondria and chloroplasts. So far, dimerization of ATP synthase has been observed only in mitochondria by biochemical and electron microscopic investigations. Although the physiological relevance remains still enigmatic, dimerization was proposed to be a unique feature of the mitochondrion [Biochim. Biophys. Acta 1555 (2002) 154]. It is hard to imagine, however, that closely related protein complexes of mitochondria and chloroplast should show such severe differences in structural organization. We present the first evidences for dimerization of chloroplast ATP synthases within the thylakoid membrane.By investigation of the thylakoid membrane of Chlamydomonas reinhardtii by blue-native polyacrylamide gel electrophoresis, dimerization of the chloroplast ATP synthase was detected. Chloroplast ATP synthase dimer dissociates into monomers upon incubation with vanadate or phosphate but not by incubation with molybdate, while the mitochondrial dimer is not affected by the incubation. This suggests a distinct dimerization mechanism for mitochondrial and chloroplast ATP synthase. Since vanadate and phosphate bind to the active sites, contact sites located on the hydrophilic CF1 part are suggested for the chloroplast ATP synthase dimer. As the degree of dimerization varies with phosphate concentration, dimerization might be a response to low phosphate concentrations.
Keywords: Chloroplast ATP synthase; Blue-native electrophoresis; Chlamydomonas reinhardtii; Membrane protein complex; Thylakoid membrane;

Higher plant-like subunit composition of mitochondrial complex I from Chlamydomonas reinhardtii: 31 conserved components among eukaryotes by Pierre Cardol; Frank Vanrobaeys; Bart Devreese; Jozef Van Beeumen; René F. Matagne; Claire Remacle (212-224).
The rotenone-sensitive NADH:ubiquinone oxidoreductase (complex I) is the most intricate membrane-bound enzyme of the mitochondrial respiratory chain. Notably the bovine enzyme comprises up to 46 subunits, while 27 subunits could be considered as widely conserved among eukaryotic complex I. By combining proteomic and genomic approaches, we characterized the complex I composition from the unicellular green alga Chlamydomonas reinhardtii. After purification by blue-native polyacrylamide gel electrophoresis (BN-PAGE), constitutive subunits were analyzed by SDS-PAGE coupled to tandem mass spectrometry (MS) that allowed the identification of 30 proteins. We compared the known complex I components from higher plants, mammals, nematodes and fungi with this MS data set and the translated sequences from the algal genome project. This revealed that the Chlamydomonas complex I is likely composed of 42 proteins, for a total molecular mass of about 970 kDa. In addition to the 27 typical components, we have identified four new complex I subunit families (bovine ESSS, PFFD, B16.6, B12 homologues), extending the number of widely conserved eukaryote complex I components to 31. In parallel, our analysis showed that a variable number of subunits appears to be specific to each eukaryotic kingdom (animals, fungi or plants). Protein sequence divergence in these kingdom-specific sets is significant and currently we cannot exclude the possibility that homology between them exists, but has not yet been detected.
Keywords: Complex I; Chlamydomonas mitochondria; Proteomics; Bioinformatics;

Reductive titration of photosystem I and differential extinction coefficient of P700+ at 810–950 nm in leaves by Vello Oja; Irina Bichele; Katja Hüve; Bahtijor Rasulov; Agu Laisk (225-234).
We describe a method of reductive titration of photosystem I (PSI) density in leaves by generating a known amount of electrons (e) in photosystem II (PSII) and measuring the resulting change in optical signal as these electrons arrive at pre-oxidized PSI. The method complements a recently published method of oxidative titration of PSI donor side e carriers P700, plastocyanin (PC) and cytochrome f by illuminating a darkened leaf with far-red light (FRL) [V. Oja, H. Eichelmann, R.B. Peterson, B. Rasulov, A. Laisk, Decyphering the 820 nm signal: redox state of donor side and quantum yield of photosystem I in leaves, Photosynth. Res. 78 (2003) 1–15], presenting a nondestructive way for the determination of PSI density in intact leaves. Experiments were carried out on leaves of birch (Betula pendula Roth) and several other species grown outdoors. Single-turnover flashes of different quantum dose were applied to leaves illuminated with FRL, and the FRL was shuttered off immediately after the flash. The number of e generated in PSII by the flash was measured as four times O2 evolution following the flash. Reduction of the pre-oxidized P700 and PC was followed as a change in leaf transmittance using a dual-wavelength detector ED P700DW (810 minus 950 nm, H. Walz, Effeltrich, Germany). The ED P700DW signal was deconvoluted into P700+ and PC+ components using the abovementioned oxidative titration method. The P700+ component was related to the absolute number of e that reduced the P700+ to calculate the extinction coefficient. The effective differential extinction coefficient of P700+ at 810–950 nm was 0.40±0.06 (S.D.)% of transmittance change per μmol P700+ m−2 or 17.6±2.4 mM−1 cm−1. The result shows that the scattering medium of the leaf effectively increases the extinction coefficient by about two times and its variation (±14% S.D.) is mainly caused by light-scattering properties of the leaf.
Keywords: Leaf; Photosystem I; P700;

Responsibility of phosphatidylglycerol for biogenesis of the PSI complex by Norihiro Sato; Kunihiro Suda; Mikio Tsuzuki (235-243).
Phosphatidylglycerol (PG) ubiquitous in thylakoid membranes of photosynthetic organisms was previously shown to contribute to accumulation of chlorophyll through analysis of the cdsA mutant of a cyanobacterium Synechocystis sp. PCC6803 defective in PG synthesis (SNC1). Here, we characterized effects of manipulation of the PG content in thylakoid membranes of Synechocystis sp. PCC6803 on the photosystem complexes to specify roles of PG in biogenesis of thylakoid membranes. SNC1 cells with PG deprivation in vivo, together with the chlorophyll decrease, exhibited a decline not in PSII, but in PSI, at the complex level as well as the subunit levels. On the other hand, the decrease in the PSI complex was accounted for by a remarkable decrease in the PSI trimer with an increase in the monomer. These symptoms of SNC1 cells were complemented in vivo by supplementation of PG. Besides, a reduction in the PG content of thylakoid membranes isolated from the wild type in vitro on treatment with phospholipase A2 (PLA2), similar to the PG-deprivation in SNC1 in vivo, brought about a decrease in the trimer population of PSI with accumulation of the monomer. These results demonstrated that PG contributes to the synthesis and/or stability of the PSI complex for maintenance of the cellular content of chlorophyll, and also to construction of the PSI trimer from the monomer at least through stabilization of the trimerized conformation.
Keywords: Phosphatidylglycerol; Photosystem I; Photosystem II; Thylakoid membrane; Synechocystis sp. PCC6803;

The mitochondrial respiratory chain of Ustilago maydis by Oscar Juárez; Guadalupe Guerra; Federico Martínez; Juan Pablo Pardo (244-251).
Ustilago maydis mitochondria contain the four classical components of the electron transport chain (complexes I, II, III, and IV), a glycerol phosphate dehydrogenase, and two alternative elements: an external rotenone-insensitive flavone-sensitive NADH dehydrogenase (NDH-2) and an alternative oxidase (AOX). The external NDH-2 contributes as much as complex I to the NADH-dependent respiratory activity, and is not modulated by Ca2+, a regulatory mechanism described for plant NDH-2, and presumed to be a unique characteristic of the external isozyme. The AOX accounts for the 20% residual respiratory activity after inhibition of complex IV by cyanide. This residual activity depends on growth conditions, since cells grown in the presence of cyanide or antimycin A increase its proportion to about 75% of the uninhibited rate. The effect of AMP, pyruvate and DTT on AOX was studied. The activity of AOX in U. maydis cells was sensitive to AMP but not to pyruvate, which agrees with the regulatory characteristics of a fungal AOX. Interestingly, the presence of DTT during cell permeabilisation protected the enzyme against inactivation.The pathways of quinone reduction and quinol oxidation lack an additive behavior. This is consistent with the competition of the respiratory components of each pathway for the quinol/quinone pool.
Keywords: Alternative NADH dehydrogenase; Alternative oxidase; Basidiomycete; Digitonin; Electron transport chain; Salicylhydroxamic acid; Permeabilisation; Ustilago maydis;

Energetics of wild-type and mutant multidrug resistance secondary transporter LmrP of Lactococcus lactis by Piotr Mazurkiewicz; Arnold J.M. Driessen; Wil N. Konings (252-261).
LmrP, a proton/multidrug antiporter of Lactococcus lactis, transports a variety of cationic substrates. Previously, two membrane-embedded acidic residues, Asp142 and Glu327, have been reported to be important for multidrug transport activity of LmrP. Here we show that neither Glu327 nor Asp142 is essential for ethidium binding but that Glu327 is a critical residue for the high affinity binding of Hoechst 33342. Substitution of these two residues, however, negatively influences the transport activity. The energetics of transport was studied of two closely related cationic substrates ethidium and propidium that carry one and two positive charges, respectively. Extrusion of monovalent ethidium is dependent on both the electrical membrane potential (Δψ) and transmembrane proton gradient (ΔpH), while extrusion of propidium predominantly depends on the ΔpH only. The LmrP mutants D142C and E327C, however, mediate electroneutral ethidium extrusion, but are unable to mediate ΔpH-dependent extrusion of propidium. These data indicate that Asp142 and Glu327 are involved in proton translocation.
Keywords: Energetics; LmrP; Lactococcus lactis;

Author Index (263-264).

Cumulative Contents (265-266).