BBA - Bioenergetics (v.1858, #7)

Nitric oxide inhibits the mitochondrial carnitine/acylcarnitine carrier through reversible S-nitrosylation of cysteine 136 by Annamaria Tonazzi; Nicola Giangregorio; Lara Console; Annalisa De Palma; Cesare Indiveri (475-482).
S-nitrosylation of the mitochondrial carnitine/acylcarnitine transporter (CACT) has been investigated on the native and the recombinant proteins reconstituted in proteoliposomes, and on intact mitochondria. The widely-used NO-releasing compound, GSNO, strongly inhibited the antiport measured in proteoliposomes reconstituted with the native CACT from rat liver mitochondria or the recombinant rat CACT over-expressed in E. coli. Inhibition was reversed by the reducing agent dithioerythritol, indicating a reaction mechanism based on nitrosylation of Cys residues of the CACT. The half inhibition constant (IC50) was very similar for the native and recombinant proteins, i.e., 74 and 71 μM, respectively. The inhibition resulted to be competitive with respect the substrate, carnitine. NO competed also with NEM, correlating well with previous data showing interference of NEM with the substrate transport path. Using a site-directed mutagenesis approach on Cys residues of the recombinant CACT, the target of NO was identified. C136 plays a major role in the reaction mechanism. The occurrence of S-nitrosylation was demonstrated in intact mitochondria after treatment with GSNO, immunoprecipitation and immunostaining of CACT with a specific anti NO-Cys antibody. In parallel samples, transport activity of CACT measured in intact mitochondria, was strongly inhibited after GSNO treatment. The possible physiological and pathological implications of the post-translational modification of CACT are discussed.
Keywords: Membrane transport; Liposome; β-Oxidation; Carnitine; Mitochondria;

Fast turnover of ferredoxin/Fd reduction by photosystem-I/PSI requires that it dissociates rapidly after it has been reduced by PSI:Fd intracomplex electron transfer. The rate constants of Fd dissociation from PSI have been determined by flash-absorption spectroscopy with different combinations of cyanobacterial PSIs and Fds, and different redox states of Fd and of the terminal PSI acceptor (FAFB). Newly obtained values were derived firstly from the fact that the dissociation constant between PSI and redox-inactive gallium-substituted Fd increases upon (FAFB) reduction and secondly from the characterization and elucidation of a kinetic phase following intracomplex Fd reduction to binding of oxidized Fd to PSI, a process which is rate-limited by the foregoing dissociation of reduced Fd from PSI. By reference to the complex with oxidized partners, dissociation rate constants were found to increase moderately with (FAFB) single reduction and by about one order of magnitude after electron transfer from (FAFB) to Fd, therefore favoring turnover of Fd reduction by PSI. With Thermosynechococcus elongatus partners, values of 270, 730 and > 10000 s−1 were thus determined for (FAFB)Fdoxidized, (FAFB)  Fdoxidized and (FAFB)Fdreduced, respectively. Moreover, assuming a conservative upper limit for the association rate constant between reduced Fd and PSI, a significant negative shift of the Fd midpoint potential upon binding to PSI has been calculated (< −60 mV for Thermosynechococcus elongatus). From the present state of knowledge, the question is still open whether this redox shift is compatible with a large (> 10) equilibrium constant for intracomplex reduction of Fd from (FAFB).
Keywords: Photosynthesis; Electron transfer; Redox potential; Ferredoxin binding; Gallium-substituted ferredoxin; Association and dissociation kinetics; Binding-induced shift of midpoint potential;

Near-neighbor interactions of the membrane-embedded subunits of the mitochondrial ATP synthase of a chlorophycean alga by Lorenzo Sánchez-Vásquez; Miriam Vázquez-Acevedo; Javier de la Mora; Félix Vega-deLuna; Pierre Cardol; Claire Remacle; Georges Dreyfus; Diego González-Halphen (497-509).
Mitochondrial F1FO-ATP synthase of the chlorophycean algae Polytomella sp. can be isolated as a highly stable dimeric complex of 1600 kDa. It is composed of eight highly conserved orthodox subunits (α, β, γ, δ, ε, OSCP, a, and c) and nine subunits (Asa1-9) that are exclusive of chlorophycean algae. The Asa subunits replace those that build up the peripheral stalk and the dimerization domains of the ATP synthase in other organisms. Little is known about the disposition of subunits Asa6, Asa8 and Asa9, that are predicted to have transmembrane stretches and that along with subunit a and a ring of c-subunits, seem to constitute the membrane-embedded Fo domain of the algal ATP synthase. Here, we over-expressed and purified the three Asa hydrophobic subunits and explored their interactions in vitro using a combination of immunochemical techniques, affinity chromatography, and an in vivo yeast-two hybrid assays. The results obtained suggest the following interactions Asa6–Asa6, Asa6–Asa8, Asa6–Asa9, Asa8–Asa8 and Asa8–Asa9. Cross-linking experiments carried out with the intact enzyme corroborated some of these interactions. Based on these results, we propose a model of the disposition of these hydrophobic subunits in the membrane-embedded sector of the algal ATP synthase. We also propose based on sequence analysis and hydrophobicity plots, that the algal subunit a is atypical in as much it lacks the first transmembrane stretch, exhibiting only four hydrophobic, tilted alpha helices.
Keywords: F1FO-ATP synthase peripheral arm; Membrane domain of the ATP synthase; Chlorophycean algae; Chlamydomonas reinhardtii; Polytomella sp.; Asa subunits;

Zeaxanthin and echinenone modify the structure of photosystem I trimer in Synechocystis sp. PCC 6803 by Sindhujaa Vajravel; Mihály Kis; Kinga Kłodawska; Hajnalka Laczko-Dobos; Przemysław Malec; László Kovács; Zoltán Gombos; Tunde N. Toth (510-518).
The function of xanthophylls in the organisation and structure of the photosynthetic complexes is not completely clarified yet. Recently, we observed a reduced level of the photosystem oligomers upon xanthophyll deficiency, although xanthophylls are not considered to be part of the photosynthetic complexes of cyanobacteria. The present study aimed at further investigating the relationship between xanthophylls and photosytem I (PSI) complex in the cyanobacterium Synechocystis sp. PCC 6803. Interestingly, we recorded the presence of echinenone and zeaxanthin in the isolated PSI trimers. These two xanthophyll species are among the most abundant xanthophylls in this cyanobacterial species. Various xanthophyll biosynthesis mutants were used to investigate the specific role of these xanthophylls. Our spectroscopic results revealed specific structural changes manifested in altered pigment-pigment or pigment-protein interactions within PSI complex in the absence of zeaxanthin and echinenone. These structural modifications of the complexes seem to destabilize the PSI trimeric complexes and eventually result in an increased propensity for monomerization. Our results clearly demonstrate that xanthophylls are important for the fine-tuning of the PSI trimer structure. These xanthophylls could be part of the complex or be embedded in the membrane in the vicinity of PSI.Display Omitted
Keywords: Carotenoid; Cyanobacteria; Echinenone; Photosynthesis; Photosystem I; Zeaxanthin;

Mitochondrial LON protease-dependent degradation of cytochrome c oxidase subunits under hypoxia and myocardial ischemia by Naresh B.V. Sepuri; Rajesh Angireddy; Satish Srinivasan; Manti Guha; Joseph Spear; Bin Lu; Hindupur K. Anandatheerthavarada; Carolyn K. Suzuki; Narayan G. Avadhani (519-528).
The mitochondrial ATP dependent matrix protease, Lon, is involved in the maintenance of mitochondrial DNA nucleoids and degradation of abnormal or misfolded proteins. The Lon protease regulates mitochondrial Tfam (mitochondrial transcription factor A) level and thus modulates mitochondrial DNA (mtDNA) content. We have previously shown that hypoxic stress induces the PKA-dependent phosphorylation of cytochrome c oxidase (CcO) subunits I, IVi1, and Vb and a time-dependent reduction of these subunits in RAW 264.7 murine macrophages subjected to hypoxia and rabbit hearts subjected to ischemia/reperfusion. Here, we show that Lon is involved in the preferential turnover of phosphorylated CcO subunits under hypoxic/ischemic stress. Induction of Lon protease occurs at 6 to 12 h of hypoxia and this increase coincides with lower CcO subunit contents. Over-expression of flag-tagged wild type and phosphorylation site mutant Vb and IVi1 subunits (S40A and T52A, respectively) caused marked degradation of wild type protein under hypoxia while the mutant proteins were relatively resistant. Furthermore, the recombinant purified Lon protease degraded the phosphorylated IVi1 and Vb subunits, while the phosphorylation-site mutant proteins were resistant to degradation. 3D structural modeling shows that the phosphorylation sites are exposed to the matrix compartment, accessible to matrix PKA and Lon protease. Hypoxic stress did not alter CcO subunit levels in Lon depleted cells, confirming its role in CcO turnover. Our results therefore suggest that Lon preferentially degrades the phosphorylated subunits of CcO and plays a role in the regulation of CcO activity in hypoxia and ischemia/reperfusion injury.
Keywords: Mitochondrial LON; CcO subunits; PKA dependent phosphorylation; Hypoxia; Heart ischemia; 3D modeling;

The spectral dependence of the irreversible non-photochemical fluorescence quenching associated with photoinhibition in vitro has been comparatively investigated in thylakoid membranes, PSII enriched particles and PSII core complexes isolated from spinach. The analysis of the fluorescence emission spectra of dark-adapted and quenched samples as a function of the detection temperature in the 280–80 K interval, indicates that Chlorophyll spectral forms having maximal emission in the 700–702 nm and 705–710 nm ranges gain relative intensity in concomitance with the establishment of irreversible light-induced quenching, acting thereby as spectroscopic markers. The relative enhancement of the 700–702 nm and 705–710 nm forms emission could be due either to an increase of their stoichiometric abundance or to their intrinsically low fluorescence quantum yields. These two factors, that can also coexist, need to be promoted by light-induced alterations in chromophore-protein as well as chromophore-chromophore interactions. The bands centred at about 701 and 706 nm are also observed in the PSII core complex, suggesting their, at least partial, localisation in proximity to the reaction centre, and the occurrence of light-induced conformational changes in the core subunits.
Keywords: Non-photochemical quenching; Irreversible light-induced quenching; Photosystem II; Core complex; Fluorescence Emission;

Photoprotection strategies of the alga Nannochloropsis gaditana by Volha U. Chukhutsina; Rikard Fristedt; Tomas Morosinotto; Roberta Croce (544-552).
Nannochloropsis spp. are algae with high potential for biotechnological applications due to their capacity to accumulate lipids. However, little is known about their photosynthetic apparatus and acclimation/photoprotective strategies. In this work, we studied the mechanisms of non-photochemical quenching (NPQ), the fast response to high light stress, in Nannochloropsis gaditana by “locking” the cells in six different states during quenching activation and relaxation. Combining biochemical analysis with time-resolved fluorescence spectroscopy, we correlated each NPQ state with the presence of two well-known NPQ components: de-epoxidized xanthophylls and stress-related antenna proteins (LHCXs). We demonstrated that after exposure to strong light, the rapid quenching that takes place in the antennas of both photosystems was associated with the presence of LHCXs. At later stages, quenching occurs mainly in the antennas of PSII and correlates with the amount of de-epoxidised xanthophylls. We also observed changes in the distribution of excitation energy between photosystems, which suggests redistribution of excitation between photosystems as part of the photo-protective strategy. A multistep model for NPQ induction and relaxation in N. gaditana is discussed.
Keywords: Heterokonta; Nannochloropsis; Photosynthesis; Non-photochemical quenching; Xanthophyll cycle;