BBA - Bioenergetics (v.1608, #2-3)

The photosystem II (PSII) complex is located in the thylakoid membrane of higher plants, algae and cyanobacteria and drives the water oxidation process of photosynthesis, which splits water into reducing equivalents and molecular oxygen by solar energy. Electron and X-ray crystallography analyses have revealed that the PSII core complex contains between 34 and 36 transmembrane α-helices, depending on the organism. Of these helices at least 12–14 are attributed to low molecular mass proteins. However, to date, at least 18 low molecular mass (<10 kDa) subunits are putatively associated with the PSII complex. Most of them contain a single transmembrane span and their protein sequences are conserved among photosynthetic organisms. In addition, these proteins do not have any similarity to any known functional proteins in any type of organism, and only two of them bind a cofactor. These findings raise intriguing questions about why there are so many small protein subunits with single-transmembrane spans in the PSII complex, and their possible functions. This article reviews our current knowledge of this group of proteins. Deletion mutations of the low molecular mass subunits from both prokaryotic and eukaryotic model systems are compared in an attempt to understand the function of these proteins. From these comparisons it seems that the majority of them are involved in stabilization, assembly or dimerization of the PSII complex. The small proteins may facilitate fast dynamic conformational changes that the PSII complex needs to perform an optimal photosynthetic activity.
Keywords: Small protein; PS2; Function; Arabidopsis; Synechocystis;

Rhodoquinone reaction site of mitochondrial complex I, in parasitic helminth, Ascaris suum by Tetsuo Yamashita; Takara Ino; Hideto Miyoshi; Kimitoshi Sakamoto; Arihiro Osanai; Eiko Nakamaru-Ogiso; Kiyoshi Kita (97-103).
The components and organization of the respiratory chain in helminth mitochondria vary remarkably depending upon the stage of the life cycle. Mitochondrial complex I in the parasitic helminth Ascaris suum uses ubiquinone-9 (UQ9) and rhodoquinone-9 (RQ9) under aerobic and anaerobic conditions, respectively. In this study, we investigated structural features of the quinone reduction site of A. suum complex I using a series of quinazoline-type inhibitors and also by the kinetic analysis of rhodoquinone-2 (RQ2) and ubiquinone-2 (UQ2) reduction. Structure–activity profiles of the inhibition by quinazolines were comparable, but not completely identical, between NADH-RQ2 and NADH-UQ2 oxidoreductase activities. However, the inhibitory mechanism of quinazolines was competitive and partially competitive against RQ2 and UQ2, respectively. The pH profiles of both activities differed remarkably; NADH-RQ2 oxidoreductase activity showed an optimum pH at 7.6, whereas NADH-UQ2 oxidoreductase activity showed two optima pH at 6.4 and 7.2. Our results indicate that although A. suum complex I uses both RQ2 and UQ2 as an electron acceptor, the manner of reaction (or binding) of the two quinones differs.
Keywords: Ascaris suum; Complex I; Rhodoquinone; Ubiquinone; Quinone reaction site;

Two separate pathways for d-lactate oxidation by Saccharomyces cerevisiae mitochondria which differ in energy production and carrier involvement by Maria Luigia Pallotta; Daniela Valenti; Michelina Iacovino; Salvatore Passarella (104-113).
The d-lactate carriers and d-lactate dehydrogenases could account for the removal of the toxic methylglyoxal from cytosol, as well as for the d-lactate-dependent gluconeogenesis.
Keywords: Saccharomyces cerevisiae; Mitochondrion; d-Lactate; Dehydrogenase; Transport; Carrier;

Light-induced excitation energy redistribution in Spirulina platensis cells: “spillover” or “mobile PBSs”? by Donghui Li; Jie Xie; Jingquan Zhao; Andong Xia; Donghai Li; Yandao Gong (114-121).
State transitions induced by light and redox were investigated by observing the 77 K fluorescence spectra for the intact cells of Spirulina platensis. To clarify if phycobilisomes (PBSs) take part in the state transition, the contributions of PBSs to light-induced state transition were studied in untreated cells and the cells treated by betaine which fixed PBSs firmly on the thylakoid membranes. It was observed that the betaine-treated cells did not show any light-induced state transition. This result definitely confirmed that the light-induced excitation energy regulation between the two photosystems is mainly dependent on a spatial movement of PBSs on the thylakoid membranes, which makes PBS cores partially decoupled from photosystem II (PSII) while PBS rods more strongly coupled with photosystem I (PSI) during the transition from state 1 to state 2. On the other hand, an energy exchange between the two photosystems was observed in both untreated and betaine-treated cells during redox-induced state transition. These observations suggested that two different mechanisms were involved in the light-induced state transition and the redox-induced one. The former involves only a physical movement of PBSs, while the latter involves not only the movement of PBS but also energy spillover from PSII to PSI. A model for light-induced state transition was proposed based on the current results as well as well known knowledge.
Keywords: C-PC; APC; PSI; PSII; State transition; Energy transfer;

Alkylsulfonates activate the uncoupling protein UCP1: implications for the transport mechanism by Eduardo Rial; Ekaitz Aguirregoitia; Jesús Jiménez-Jiménez; Amalia Ledesma (122-130).
Fatty acids activate the uncoupling protein UCP1 by a still controversial mechanism. Two models have been put forward where the fatty acid operates as either substrate (“fatty acid cycling hypothesis”) or prosthetic group (“proton buffering model”). Two sets of experiments that should help to discriminate between the two hypothetical mechanisms are presented. We show that undecanosulfonate activates UCP1 in respiring mitochondria under conditions identical to those required for the activation by fatty acids. Since alkylsulfonates cannot cross the lipid bilayer, these experiments rule out the fatty acid cycling hypothesis as the mechanism of uncoupling. We also demonstrate that without added nucleotides and upon careful removal of endogenous fatty acids, brown adipose tissue (BAT) mitochondria from cold-adapted hamsters respire at the full uncoupled rate. Addition of nucleotides lower the respiratory rate tenfold. The high activity observed in the absence of the two regulatory ligands is an indication that UCP1 displays an intrinsic proton conductance that is fatty acid-independent. We propose that the fatty acid uncoupling mediated by other members of the mitochondrial transporter family probably involves a carrier to pore transition and therefore has little in common with the activation of UCP1.
Keywords: UCP1; Uncoupling protein; Mitochondria; Fatty acid; Brown adipose tissue; Permeability transition;

A mathematical model is proposed showing that the mono-exponential recovery of phosphocreatine (PCr) after exercise is an approximation of a more complex pattern, which is identified by a second-order differential equation. The model predicts the possibility of three different patterns of PCr recovery: bi-exponential, oscillatory damped, and critically damped; the mono-exponential pattern being a particular case of the functions which are solutions of the differential equation. The model was tested on a sample of recovery data from 50 volunteers, checking whether the recovery patterns predicted by the model lead to a significant improvement of fit (IF) compared with the mono-exponential pattern. Results show that the IF is linked to pH. Bi-exponential solutions showed an IF in the pH range 6.65–6.85, and the oscillatory solutions at pH>6.9. Critically damped solutions displayed a poor IF. Oscillation frequencies found in the oscillatory recoveries increase at increasing pH. These results show that pH has a pivotal role on the pattern of PCr recovery and implications on the regulation of oxidative phosphorylation are discussed.
Keywords: PCr recovery; Cytosolic pH; Oxidative phosphorylation;

Oxidation-reduction properties of maize ferredoxin:sulfite oxidoreductase by Masakazu Hirasawa; Masato Nakayama; Toshiharu Hase; David B. Knaff (140-148).
Oxidation-reduction titrations have been carried out on the wild-type, ferredoxin-dependent sulfite reductase from maize and two site-specific variants of the enzyme. E m values have been determined for the siroheme and [4Fe–4S] cluster prosthetic groups of the enzyme, which titrate as independent, one-electron carriers. Visible-region difference spectra suggest that reduction of the [4Fe–4S] cluster significantly perturbs the spectrum of the reduced siroheme group of the enzyme. The effects of siroheme axial ligation, by either cyanide or phosphate ligands, on the redox properties of sulfite reductase have also been examined. For comparison, the effects of phosphate and cyanide on the redox properties of the ferredoxin-dependent nitrite reductase of spinach chloroplasts, an enzyme with the same prosthetic group arrangement as sulfite reductase, have been examined.
Keywords: Chloroplast sulfite reductase; Oxidation-reduction property; Chloroplast nitrite reductase; Siroheme; Iron–sulfur cluster;

Different sensitivities of CPT I and CPT II for inhibition by l-aminocarnitine in human skeletal muscle by Kathrin Traufeller; Frank Norbert Gellerich; Stephan Zierz (149-154).
l-Aminocarnitine (l-AC) has been shown to inhibit carnitine palmitoyltransferases (CPT) in rat muscle and in rat liver. However, there are no reports on interactions of l-AC with CPT II and CPT I of human muscle. Therefore, the aim of the present work was to characterize the inhibition of human muscle CPT I and CPT II by l-AC in muscle mitochondria, skinned fibers and muscle homogenates in comparison to the established action of malonyl-CoA. Both isoenzymes were inhibited by l-AC, but sensitivity was different (CPT I, K d=3.8 mM l-AC; CPT II, K d=21.3 μM l-AC). A mixed inhibition type in respect to carnitine was detected (K i=3.5 μM l-AC). At 0.5 mM l-AC, CPT II was completely inhibited without affection of CPT I. In contrast, CPT I was completely inhibited by 0.4 mM malonyl-CoA (K d=0.5 μM), whereas CPT II was nearly not affected by this inhibitor. Using these inhibitors in muscle homogenates, activities of CPT II and CPT I were detected to be 38±10% and 63±10% of total, respectively (n=21). In intact mitochondria and different fractions of muscle homogenates after selective solubilization of CPT II by Tween 20, the extent of specific CPT inhibition changed in relation to the accessible isoenzyme pattern. Palmitoyl-carnitine-dependent respiration in skinned fibers was inhibited by high concentrations of l-AC, indicating that the inhibitor can be transported via the acyl-carnitine transporter, too. The combined use of both inhibitors (l-AC and malonyl-CoA) allows the kinetic characterization of CPT I and CPT II in human muscle homogenates. In addition, it has been shown that l-AC can be used for the study of metabolic consequences of CPT II deficiency on function of intact mitochondria.
Keywords: CPT; Human muscle mitochondria; l-aminocarnitine; Inhibition;

Complex formation between ferredoxin and Synechococcus ferredoxin:nitrate oxidoreductase by Masakazu Hirasawa; Luis M. Rubio; Jeannie L. Griffin; Enrique Flores; Antonia Herrero; Jun Li; Sung-Kun Kim; John K. Hurley; Gordon Tollin; David B. Knaff (155-162).
The ferredoxin-dependent nitrate reductase from the cyanobacterium Synechococcus sp. PCC 7942 has been shown to form a high-affinity complex with ferredoxin at low ionic strength. This complex, detected by changes in both the absorbance and circular dichroism (CD) spectra, did not form at high ionic strength. When reduced ferredoxin served as the electron donor for the reduction of nitrate to nitrite, the activity of the enzyme declined markedly as the ionic strength increased. In contrast, the activity of the enzyme with reduced methyl viologen (a non-physiological electron donor) was independent of ionic strength. These results suggest that an electrostatically stabilized complex between Synechococcus nitrate reductase and ferredoxin plays an important role in the mechanism of nitrate reduction catalyzed by this enzyme. Treatment of Synechococcus nitrate reductase with either an arginine-modifying reagent or a lysine-modifying reagent inhibited the ferredoxin-dependent activity of the enzyme but did not affect the methyl viologen-dependent activity. Treatment with these reagents also resulted in a large decrease in the affinity of the enzyme for ferredoxin. Formation of a nitrate reductase complex with ferredoxin prior to treatment with either reagent protected the enzyme against loss of ferredoxin-dependent activity. These results suggest that lysine and arginine residues are present at the ferredoxin-binding site of Synechococcus nitrate reductase. Results of experiments using site-specific, charge reversal variants of the ferredoxin from the cyanobacterium Anabaena sp. PCC 7119 as an electron donor to nitrate reductase were consistent with a role for negatively charged residues on ferredoxin in the interaction with Synechococcus nitrate reductase.
Keywords: Nitrate reductase; Ferredoxin-binding; Electrostatic interaction;

Temperature and pH dependence of energy balance by 31P- and 1H-MRS in anaerobic frog muscle by Alessandra Vezzoli; Maristella Gussoni; Fulvia Greco; Lucia Zetta; Paolo Cerretelli (163-170).
The temperature (T)-dependence of energy consumption of resting anaerobic frog gastrocnemii exposed to different, changing electrochemical gradients was assessed. To this aim, the rate of ATP resynthesis (Δ∼P/Δt) was determined by 31P- and 1H-MRS as the sum of the rates of PCr hydrolysis (Δ[PCr]/Δt) and of anaerobic glycolysis (Δ[La]/ Δt, based on a ∼P/La ratio of 1.5). The investigated T levels were 15, 20 and 25 °C, whereas initial extracellular pH (pHe) values were 7.9, 7.3 and 7.0, i.e. higher, equal or lower, respectively, than intracellular pH (pHi). The latter was changing with T according to the neutrality point (dpH/dT=−0.0165 pH units/°C).Both rates of PCr hydrolysis and of lactate accumulation and that of their sum, expressed as Δ∼P/Δt, were highly T-dependent. By contrast, the pHe-dependence of the muscle energy balance was nil or extremely limited at 15 and 20 °C, respectively, but remarkable at 25 °C (with a depression of the ATP resynthesis rate up to 25% with a decrease of pHe from 7.9 to 7.0). The pHe-dependent reduction of metabolic rate was associated with a down-regulation of anaerobic glycolysis due to reduced activity of ion-transporters controlling acid–base balance and/or to a shift from Na+/H+ to a more efficient Na+-dependent Cl/HCO3 exchanger. Uncoupling of glycogenolysis from P-metabolite concentrations, both as function of T (≥20 °C) and of pHe (≤7.3), was also shown, attributable to a T-dependence of glycolytic enzyme activity and/or H+ ion transport systems.The described metabolic slowdown observed in isolated muscle preparations subjected to the combined regimes of anoxia/acidosis implies that the mechanism determining survival time at the cellular level is mediated by exchange transport systems. A similar mechanism might affect muscle metabolism of homeotherms during chronic hypoxia and/or ischemia.
Keywords: Temperature; pH; Anaerobic metabolism; MRS; Uncoupling of glycogenolysis; Frog gastrocnemius muscle;

Mixed photosystem II (PSII) samples consisting of Cl-depleted and active, or Ca2+-depleted and active PSII enriched membrane fragments, respectively, were investigated with respect to their susceptibility to light. In the presence of Cl-depleted PSII, active centers were damaged more severely, most likely caused by a higher amount of reactive oxygen species formed in the nonfunctional centers. Cl depletion led to an increased H2O2 production, which seemed to be responsible for the stimulation of PSII activity loss. To distinguish between direct H2O2 formation by partial water oxidation and indirect H2O2 formation by oxygen reduction involving the prior formation of O2 −• , the production of reactive oxygen species was followed by spin trapping EPR spectroscopy. All samples investigated, i.e. PSII with a functional water splitting complex, Ca2+- and Cl-depleted PSII, produced upon illumination O2 −• and OH radicals on the acceptor side, while Cl-depleted PSII produced additionally OH radicals originating from H2O2 formed on the donor side of PSII.
Keywords: Photosynthesis; Photosystem II; Reactive oxygen species; Photoinhibition; Spin Trapping EPR;

We have purified plant alternative oxidase (AOX) protein from the spadices of thermogenic Arum maculatum (cuckoo pint) to virtual homogeneity. The obtained enzyme fraction exhibits a high specific activity, consuming on average 32 μmol oxygen min−1 mg−1, which is completely stable for at least 6 months when the sample is stored at −70 °C. This exceptionally stable AOX activity is inhibited approximately 90% (I 50∼10 μM) by 8-hydroxyquinoline (8-OHQ) and also, although to a lesser extent, by other metal chelators such as o-phenanthroline, α,α′-dipyridyl and EDTA. When inhibited by 8-OHQ, AOX activity is fully restored upon addition of 1.2 mM ferric iron, but neither ferrous iron nor manganese has any effect, whilst zinc decreases activity even further. Furthermore, we have developed a spectrophotometric assay to measure AOX activity in an accurate manner, which will facilitate future steady state and transient kinetic studies. The reliability of this assay is evidenced by retained stability of AOX protein during the course of the reaction, reproducibility of the measured initial rates, an observed 2:1 duroquinol–oxygen stoichiometry and by the fact that, in absolute terms, the measured rates of duroquinone formation and duroquinol disappearance are identical.
Keywords: Plant mitochondrion; Alternative oxidase; Protein purification; Enzyme activation; Quinol oxidase; Diiron protein;

Roles of histidine residues in plant vacuolar H+-pyrophosphatase by Yi Y Hsiao; Ru C Van; Shu H Hung; Hsin H Lin; Rong L Pan (190-199).
Vacuolar proton pumping pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a pivotal role in electrogenic translocation of protons from cytosol to the vacuolar lumen at the expense of PPi hydrolysis. Alignment analysis on amino acid sequence demonstrates that vacuolar H+-PPase of mung bean contains six highly conserved histidine residues. Previous evidence indicated possible involvement of histidine residue(s) in enzymatic activity and H+-translocation of vacuolar H+-PPase as determined by using histidine specific modifier, diethylpyrocarbonate [J. Protein Chem. 21 (2002) 51]. In this study, we further attempted to identify the roles of histidine residues in mung bean vacuolar H+-PPase by site-directed mutagenesis. A line of mutants with histidine residues singly replaced by alanine was constructed, over-expressed in Saccharomyces cerevisiae, and then used to determine their enzymatic activities and proton translocations. Among the mutants scrutinized, only the mutation of H716 significantly decreased the enzymatic activity, the proton transport, and the coupling ratio of vacuolar H+-PPase. The enzymatic activity of H716A is relatively resistant to inhibition by diethylpyrocarbonate as compared to wild-type and other mutants, indicating that H716 is probably the target residue for the attack by this modifier. The mutation at H716 of V-PPase shifted the optimum pH value but not the T 1/2 (pretreatment temperature at which half enzymatic activity is observed) for PPi hydrolytic activity. Mutation of histidine residues obviously induced conformational changes of vacuolar H+-PPase as determined by immunoblotting analysis after limited trypsin digestion. Furthermore, mutation of these histidine residues modified the inhibitory effects of F and Na+, but not that of Ca2+. Single substitution of H704, H716 and H758 by alanine partially released the effect of K+ stimulation, indicating possible location of K+ binding in the vicinity of domains surrounding these residues.
Keywords: H+-pyrophosphatase; Tonoplast; Vacuole; Proton translocation; Histidine;