BBA - Bioenergetics (v.1556, #2-3)

Raman spectroscopy of chlorophyll d from Acaryochloris marina by Zheng-Li Cai; Heping Zeng; Min Chen; Anthony W.D Larkum (89-91).
The Raman spectroscopy of chlorophyll (Chl) d isolated from Acaryochloris marina has been measured in the range of 250–3200 cm−1 at 77 K following excitation of its Bx band at 488 nm. A peak at 1659 cm−1 of medium intensity arising from CO stretching vibration in the formyl group 31 specific to Chl d was observed clearly. Peaks due to other CO stretching vibrations of the 131 keto-, 133 ester- and 173 groups have also been observed with much weaker intensities. Intense Raman peaks in the range of 1000–1800 cm−1 are reported and homologous comparison with corresponding Raman shifts of Chl a, Chl b and BChl a are presented.
Keywords: Chlorophyll d; Acaryochloris marina; Raman spectrum; Vibration;

CP43, a component of Photosystem II (PSII) in higher plants, algae and cyanobacteria, is encoded by the psbC gene. Previous work demonstrated that alteration of an arginine residue occurring at position 305 to serine produced a strain (R305S) with altered PSII characteristics including lower oxygen-evolving activity, fewer assembled reaction centers, higher sensitivity to photoinactivation, etc. [Biochemistry 38 (1999) 1582]. Additionally, it was determined that the mutant exhibited an enhanced stability of its S2 state. Recently, we observed a significant chloride effect under chloride-limiting conditions. The mutant essentially lost the ability to grow photoautotrophically, assembled fewer fully functional PSII reaction centers and exhibited a very low rate of oxygen evolution. Thus, the observed phenotype of this mutation is very similar to that observed for the ΔpsbV mutant, which lacks cytochrome c 550 (Biochemistry 37 (1998) 1551). A His-tagged version of the R305S mutant was produced to facilitate the isolation of PSII particles. These particles were analyzed for the presence of cytochrome c 550. Reduced minus oxidized difference spectroscopy and chemiluminescence examination of Western blots indicated that cytochrome c 550 was absent in these PSII particles. Whole cell extracts from the R305S mutant, however, contained a similar amount of cytochrome c 550 to that observed in the control strain. These results indicate that the mutation R305S in CP43 prevents the strong association of cytochrome c 550 with the PSII core complex. We hypothesize that this residue is involved in the formation of the binding domain for the cytochrome.
Keywords: CP43; Oxygen evolution; Photosystem II; Chloride; Cytochrome c 550;

Opening of potassium channels modulates mitochondrial function in rat skeletal muscle by Grazyna Debska; Anna Kicinska; Jolanta Skalska; Adam Szewczyk; Rebecca May; Christian E Elger; Wolfram S Kunz (97-105).
We have investigated the presence of diazoxide- and nicorandil-activated K+ channels in rat skeletal muscle. Activation of potassium transport in the rat skeletal muscle myoblast cell line L6 caused a stimulation of cellular oxygen consumption, implying a mitochondrial effect. Working with isolated rat skeletal muscle mitochondria, both potassium channel openers (KCOs) stimulate respiration, depolarize the mitochondrial inner membrane and lead to oxidation of the mitochondrial NAD-system in a strict potassium-dependent manner. This is a strong indication for KCO-mediated stimulation of potassium transport at the mitochondrial inner membrane. Moreover, the potassium-specific effects of both diazoxide and nicorandil on oxidative phosphorylation in skeletal muscle mitochondria were completely abolished by the antidiabetic sulfonylurea derivative glibenclamide, a well-known inhibitor of ATP-regulated potassium channels (KATP channels). Since both diazoxide and nicorandil facilitated swelling of de-energised mitochondria in KSCN buffer at the same concentrations, our results implicate the presence of a mitochondrial ATP-regulated potassium channel (mitoKATP channel) in rat skeletal muscle which can modulate mitochondrial oxidative phosphorylation.
Keywords: Skeletal muscle; Mitochondrial ATP-regulated potassium channel; Potassium channel opener; Oxidative phosphorylation;

Hybrid ubiquinone: novel inhibitor of mitochondrial complex I by Hiromi Yabunaka; Atsushi Kenmochi; Yasushi Nakatogawa; Kimitoshi Sakamoto; Hideto Miyoshi (106-112).
We synthesized novel ubiquinone analogs by hybridizing the natural ubiquinone ring (2,3-dimethoxy-5-methyl-1,4-benzoquinone) and hydrophobic phenoxybenzamide unit, and named them hybrid ubiquinones (HUs). The HUs worked as electron transfer substrates with bovine heart mitochondrial succinate–ubiquinone oxidoreductase (complex II) and ubiquinol–cytochrome c oxidoreductase (complex III), but not with NADH–ubiquinone oxidoreductase (complex I). With complex I, they acted as inhibitors in a noncompetitive manner against exogenous short-chain ubiquinones irrespective of the presence of the natural ubiquinone ring. Elongation of the distance between the ubiquinone ring and the phenoxybenzamide unit did not recover the electron accepting activity. The structure/activity study showed that high structural specificity of the phenoxybenzamide moiety is required to act as a potent inhibitor of complex I. These findings indicate that binding of the HUs to complex I is mainly decided by some specific interaction of the phenoxybenzamide moiety with the enzyme. It is of interest that an analogous bulky and hydrophobic substructure can be commonly found in recently registered synthetic pesticides the action site of which is mitochondrial complex I.
Keywords: Mitochondrial complex I; Ubiquinone; Structure–activity relationship;

Kinetics of the spectral changes during reduction of the Na+-motive NADH:quinone oxidoreductase from Vibrio harveyi by Alexander V Bogachev; Yulia V Bertsova; Enno K Ruuge; Mårten Wikström; Michael I Verkhovsky (113-120).
Two radical signals with different line widths are seen in the Na+-translocating NADH:ubiquinone oxidoreductase (Na+-NQR) from Vibrio harveyi by EPR spectroscopy. The first radical is observed in the oxidized enzyme, and is assigned as a neutral flavosemiquinone. The second radical is observed in the reduced enzyme and is assigned to be the anionic form of flavosemiquinone. The time course of Na+-NQR reduction by NADH, as monitored by stopped-flow optical spectroscopy, shows three distinct phases, the spectra of which suggest that they correspond to the reduction of three different flavin species. The first phase is fast both in the presence and absence of sodium, and is assigned to reduction of FAD to FADH2 at the NADH dehydrogenating site. The rates of the other two phases are strongly dependent on sodium concentration, and these phases are attributed to reduction of two covalently bound FMN's. Combination of the optical and EPR data suggests that a neutral FMN flavosemiquinone preexists in the oxidized enzyme, and that it is reduced to the fully reduced flavin by NADH. The other FMN moiety is initially oxidized, and is reduced to the anionic flavosemiquinone. One-electron transitions of two discrete flavin species are thus assigned as sodium-dependent steps in the catalytic cycle of Na+-NQR.
Keywords: Na+-NADH:quinone oxidoreductase; Flavin cofactor; Na+-pump; Respiratory enzyme; Bacteria; Vibrio harveyi;

Nicotinamide adenine dinucleotide—reduced form (NADH):quinone oxidoreductase (respiratory Complex I), F420H2 oxidoreductase and complex, membrane-bound NiFe-hydrogenase contain protein subunits homologous to a certain type of bona fide antiporters. In Complex I, these polypeptides (NuoL/ND5, NuoM/ND4, NuoN/ND2) are most likely core components of the proton pumping mechanism, and it is thus important to learn more about their structure and function. In this work, we have determined the transmembrane topology of one such polypeptide, and built a 2D structural model of the protein valid for all the homologous polypeptides. The experimentally determined transmembrane topology was different from that predicted by majority vote hydrophobicity analyses of members of the superfamily. A detailed phylogenetic analysis of a large set of primary sequences shed light on the functional relatedness of these polypeptides.
Keywords: NADH:quinone oxidoreductase; F420H2 oxidoreductase; NiFe-hydrogenase; Antiporter; Fusion protein; Alkaline phosphatase;

Dimerization of F0F1ATP synthase from bovine heart is independent from the binding of the inhibitor protein IF1 by Lara Tomasetig; Francesca Di Pancrazio; David A Harris; Irene Mavelli; Giovanna Lippe (133-141).
Solubilization of heavy bovine heart mitochondria with Triton X-100 leads to the selective extraction of F0F1ATP synthase monomer and dimer in a 2:1 ratio, as revealed by blue native gel electrophoresis (BN-PAGE). Second dimensional SDS-PAGE and immunoblotting with IF1 and F1 antibodies following BN-PAGE show that both aggregation states of the ATP synthase contain IF1. The monomer/dimer ratio does not change in extracts from mitochondria subjected to different energy conditions accompanied by IF1 binding modulation or from submitochondrial particles differing in IF1 content. In addition, the usual monomer/dimer ratio is observed even in submitochondrial particles deprived of IF1. Histochemical staining for ATPase activity demonstrates that the dimer is inactive, irrespective of its IF1 content. It is concluded that in the membrane of bovine heart mitochondria the ATP synthase dimer is a stable inactive structure, whose formation is not mediated by IF1 binding.
Keywords: Mitochondrial F0F1ATPsynthase; Dimerization; Inhibitor protein IF1; Blue native polyacrylamide electrophoresis; Histochemical staining;

Expression of chaA, a sodium ion extrusion system of Escherichia coli, is regulated by osmolarity and pH by Toshiaki Shijuku; Takafumi Yamashino; Hyuma Ohashi; Hiromi Saito; Tomohito Kakegawa; Michio Ohta; Hiroshi Kobayashi (142-148).
ChaA, one of the sodium ion extrusion systems of Escherichia coli, was found to function at high pH [Biochim. Biophys. Acta 1363 (1998) 231]. A chaAlacZ transcriptional fusion gene was constructed using chaA of E. coli O157:H7 and its expression was observed in strains derived from E. coli K12. The fusion gene was expressed at high pH and was induced by the addition of NaCl, KCl or sucrose. The amount of chaA mRNA measured by reverse transcription-polymerase chain reaction (RT-PCR) was increased by the addition of sucrose to alkaline growth medium. These results suggested that chaA expression was regulated by medium osmolarity and pH.
Keywords: Antiporter; Na+/H+; Gene expression; chaA; Escherichia coli;

Squalane is in the midplane of the lipid bilayer: implications for its function as a proton permeability barrier by Thomas Hauß; Silvia Dante; Norbert A Dencher; Thomas H Haines (149-154).
A recently proposed model for proton leakage across biological membranes [Prog. Lipid Res. 40 (2001) 299] suggested that hydrocarbons specifically in the center of the lipid bilayer inhibit proton leaks. Since cellular membranes maintain a proton electrochemical gradient as a principal energy transducer, proton leakage unproductively consumes cellular energy. Hydrocarbons in the bilayer are widespread in membranes that sustain such gradients. The alkaliphiles are unique in that they contain up to 40 mol% isoprenes in their membranes including 10–11 mol% squalene [J. Bacteriol. 168 (1986) 334]. Squalene is a polyisoprene hydrocarbon without polar groups. Localizing hydrocarbons in lipid bilayers has not been trivial. A myriad of physical methods including fluorescence spectroscopy, electron-spin resonance, nuclear magnetic resonance as well as X-ray and neutron diffraction have been used to explore this question with various degrees of success and often contradictory results. Seeking unambiguous evidence for the localization of squalene in membranes or lipid bilayers, we employed neutron diffraction. We incorporated 10 mol% perdeuterated or protonated squalane, an isosteric analogue of squalene, into stacked bilayers of dioleoyl phosphatidyl choline (DOPC) doped with dioleoyl phosphatidyl glycerol (DOPG) to simulate the negative charges found on natural membranes. The neutron diffraction data clearly show that the squalane lies predominantly in the bilayer center, parallel to the plane of the membrane.
Keywords: Proton leak; Squalene; Squalane; Proton gradient; Ubiquinone; Neutron diffraction; Lipid bilayer;

Bax and heart mitochondria: uncoupling and inhibition of respiration without permeability transition by Florence Appaix; Karen Guerrero; David Rampal; Mohamed Izikki; Tuuli Kaambre; Peeter Sikk; Dieter Brdiczka; Catherine Riva-Lavieille; Jose Olivares; Michel Longuet; Bruno Antonsson; Valdur A Saks (155-167).
The effects of Bax (full-length, FL, and C-terminal truncated, ΔC) on respiration rate, membrane potential, MgATPase activity and kinetics of regulation of respiration were studied in isolated rat heart mitochondria and permeabilized cardiomyocytes. The results showed that while both Bax-FL and Bax-ΔC permeabilized the outer mitochondrial membrane, released cytochrome c and reduced the respiration rate, the latter could be fully restored by exogenous cytochrome c only in the case of Bax-ΔC, but not in presence of Bax-FL. In addition, Bax-FL but not Bax-ΔC increased the MgATPase activity, and their effects on the mitochondrial membrane potential were quantitatively different. None of these effects was sensitive to cyclosporin A (CsA).It is concluded that Bax-FL affects both the outer and the inner mitochondrial membranes by: (1) opening large pores in the outer membrane; (2) inhibiting some segments of the respiratory chain in the inner membrane; and (3) uncoupling the inner mitochondrial membrane by increasing proton leak without opening the permeability transition pore (PTP).
Keywords: Mitochondrion; Apoptosis; Heart; Oxidative phosphorylation; Bax;

The crystal structure of the light-harvesting phycobiliprotein, c-phycocyanin from the thermophilic cyanobacterium Synechococcus vulcanus has been refined to 1.6 Å resolution based on the previously determined lower resolution structure (PDB entry 1I7Y). The improved data was collected using synchrotron radiation at 100 K. The significantly improved crystallographic data has lead to improved calculated electron density maps, allowing the unambiguous positioning of all protein and co-factor atoms and the positioning of 377 solvent molecules. The positions of solvent molecules at specific sites important for stabilization of different levels of self-assembly of the phycobilisome structure were identified and the bonding network is described. The presence of solvent molecules in the vicinity of the co-factors and in intermolecular spaces is identified and their possible roles are suggested. All three of the phycocyanobilin co-factors bind water molecules at specific sites between the propionic acid side chains. Molecular dynamic (MD) simulations support that these special waters have a role in stabilization of this conformation. On the basis of the crystal packing reported here and in comparison to other phycobiliprotein crystal forms, we have analyzed the roles of specific sites on the formation of the phycobilisome complex.
Keywords: Photosynthesis; Antenna protein; Photosystem II; Energy transfer;

Spectroscopic and oxidation–reduction properties of Rhodobacter capsulatus cytochrome c 1 and its M183K and M183H variants by Jun Li; Elisabeth Darrouzet; Ish K Dhawan; Michael K Johnson; Artur Osyczka; Fevzi Daldal; David B Knaff (175-186).
Two variants of the cytochrome c 1 component of the Rhodobacter capsulatus cytochrome bc 1 complex, in which Met183 (an axial heme ligand) was replaced by lysine (M183K) or histidine (M183H), have been analyzed. Electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) spectra of the intact complex indicate that the histidine/methionine heme ligation of the wild-type cytochrome is replaced by histidine/lysine ligation in M183K and histidine/histidine ligation in M183H. Variable amounts of histidine/histidine axial heme ligation were also detected in purified wild-type cytochrome c 1 and its M183K variant, suggesting that a histidine outside the CSACH heme-binding domain can be recruited as an alternative ligand. Oxidation–reduction titrations of the heme in purified cytochrome c 1 revealed multiple redox forms. Titrations of the purified cytochrome carried out in the oxidative or reductive direction differ. In contrast, titrations of cytochrome c 1 in the intact bc 1 complex and in a subcomplex missing the Rieske iron–sulfur protein were fully reversible. An E m7 value of −330 mV was measured for the single disulfide bond in cytochrome c 1. The origins of heme redox heterogeneity, and of the differences between reductive and oxidative heme titrations, are discussed in terms of conformational changes and the role of the disulfide in maintaining the native structure of cytochrome c 1.
Keywords: Rhodobacter capsulatus cytochrome c 1; Axial heme ligand; Oxidation–reduction titration; EPR and MCD spectroscopy; Disulfide bond;

Genistein, a natural isoflavone present in soybeans, is a potent agent in the prophylaxis and treatment of cancer. Addition of genistein to isolated rat liver mitochondria (RLM) induces swelling, loss of membrane potential and release of accumulated Ca2+. These changes are Ca2+-dependent and are prevented by cyclosporin A (CsA) and bongkrekic acid (BKA), two classical inhibitors of the mitochondrial permeability transition (MPT). Induction of the MPT by genistein is accompanied by oxidation of thiol groups and pyridine nucleotides. The reducing agent dithioerythritol and the alkylating agent N-ethylmaleimide (NEM) completely prevent the opening of the transition pore, thereby emphasizing that the effect of the isoflavone correlates with the mitochondrial redox state. Further analyses showed that genistein induces the MPT by the generation of reactive oxygen species (ROS) due to its interaction with the respiratory chain at the level of mitochondrial complex III.
Keywords: Genistein; Mitochondria; Electron transport chain; Permeability transition;

Relevance of the diastereotopic ligation of magnesium atoms of chlorophylls in Photosystem I by Teodor Silviu Balaban; Petra Fromme; Alfred R Holzwarth; Norbert Krauß; Valentin I Prokhorenko (197-207).
The central magnesium (Mg) atoms of natural occurring tetrapyrroles such as chlorophylls (Chls) and bacteriochlorophylls (BChls) are typically five-coordinated, a fact which leads to the formation of diastereoisomers if the Mg–ligand bond is stable on the time scale of the observation method. This possibility has only been briefly addressed before in a CD-study of BChl c aggregates [T.S. Balaban, A.R. Holzwarth, K. Schaffner, J. Mol. Struct. 349 (1995) 183]. On the basis of the chlorophyll–protein complex photosystem I (PSI), which has recently been characterized by single crystal crystallography [P. Jordan, P. Fromme, H.T. Witt, O. Klukas, W. Saenger, N. Krauß, Nature 411 (2001) 909], we find that chlorophyll a molecules are much more frequently bound by the protein matrix from one side (anti) than the other one (syn) in a ratio of 82:14, which corresponds to a significant ΔΔG value of 4.3 kJ/mol. Syn and anti denote the orientation of the Mg–ligand with respect to the 17-propionic acid esterified by phytol. Furthermore, by parallel sequence analysis we find that the binding sites for both syn and anti chlorophylls have been strongly conserved during evolution—a fact which stresses the nonrandom manner in which chlorophylls are bound by the apoprotein in antenna complexes, in order to exert efficiently their light harvesting function and energy funnelling. Most remarkably, all the syn chlorophylls are part of the inner core antenna system. Results from semiempirical quantum mechanical and detailed exciton coupling calculations allow us to speculate on the functional relevance of the diasteretopicity for PSI functioning.
Keywords: Crystal structure; Photosystem I; Sequence alignment; Diastereotopicity; Chlorophyll complex;

The H+-ATP synthase from chloroplasts, CF0F1, was isolated, reconstituted into liposomes and ATP synthesis activity was measured after energization of the proteoliposomes with an acid–base transition. The ATP yield was measured as a function of the reaction time after energization, the data were fitted by an exponential function and the initial rate was calculated from the fit parameters. CF0F1 was reconstituted by detergent dialysis in asolectin liposomes and phosphatidylcholine/phosphatidic acid (PtdCho/PtdAc from egg yolk) liposomes. In asolectin liposomes, high initial rates of ATP synthesis (up to 400 s−1) were observed with a rapid decline of the rate; in PtdCho/PtdAc liposomes the initial rate is smaller (up to 200 s−1), but the decline of the activity is slower. CF0F1 was reconstituted into PtdCho/PtdAc liposomes either by detergent dialysis or into reverse phase liposomes. The dependence of the rate of ATP synthesis on the phosphate concentration was measured with both types of proteoliposomes. The data can be described by Michaelis–Menten kinetics with a K M value of 350 μM for reverse phase liposomes and a K M value of 970 μM for dialysis liposomes. Both K M values depend neither on the magnitude of ΔpH nor on the electric potential difference, whereas V max decreases strongly with decreasing energization. At low phosphate concentration, there are small deviations from Michaelis–Menten kinetics. The measured rates are higher than those calculated from the fitted Michaelis–Menten parameters. This effect is interpreted as evidence that more than one phosphate binding site is involved in ATP synthesis.
Keywords: Phosphate; ATP; Chloroplast;

The NADP-reducing hydrogenase from Desulfovibrio fructosovorans: functional interaction between the C-terminal region of HndA and the N-terminal region of HndD subunits by Zorah Dermoun; Gilles De Luca; Marcel Asso; Patrick Bertrand; Françoise Guerlesquin; Bruno Guigliarelli (217-225).
The hndABCD operon from Desulfovibrio fructosovorans encodes an uncommon heterotetrameric NADP-reducing iron hydrogenase. The presence of a [2Fe–2S] cluster likely located in the C-terminal region of the HndA subunit has already been revealed. We have cloned and expressed the truncated hndA gene in Escherichia coli to isolate the structural [2Fe–2S] module. Optical and EPR spectra are found identical to that of the native HndA subunit and the midpoint redox potential (−385 mV) is similar to that of the native protein (−395 mV). These results clearly demonstrate that the C-terminal region of HndA is a structurally independent [2Fe2S] ferredoxin-like domain. In the same way, the N-terminal domain of the HndD subunit was overproduced in E. coli and characterized. The presence of a [2Fe–2S] cluster was evidenced by optical spectroscopy. The midpoint redox potential (−380 mV) of this domain was found very close to that of the truncated HndA subunit but the EPR properties were significantly different. The various EPR properties allowed us to observe an electron exchange between the two [2Fe–2S] ferredoxin-like domains of the HndA and HndD subunits. Moreover, domain–domain interactions, observed by far-western experiments, indicate that these subunits are direct partners in the native complex.
Keywords: Desulfovibrio; Electron transfer; [2Fe–2S] domain; EPR; NADP-reducing hydrogenase;

Recent X-ray crystallographic analyses of the mitochondrial cytochrome bc 1 complex show ubiquinone binding at the Qi site, but attempts to show binding of ubiquinol or ubiquinone at the Qo site have been unsuccessful, even though the binding of noncompetitive Qo site inhibitors near the putative ubiquinol binding pocket is well established. We speculate that ubiquinol binds transiently to the Qo site only when both heme bL and the iron sulfur cluster are in the oxidized form, an experimental condition difficult to obtain since ubiquinol will be oxidized once bound to the site. Stable binding at the Qo site might be achieved by a nonoxidizable ubiquinol-like compound. For this purpose, the isomers 2,3,4-trimethoxy-5-decyl-6-methyl-phenol (TMDMP) and 2,3,4-trimethoxy-5-methyl-6-decyl-phenol (TMMDP) were synthesized from 2,3-dimethoxy-5-methyl-6-decyl-1, 4-benzoquinol (Q0C10) by controlled methylation and separated by TLC and HPLC. The structures of TMDMP and TMMDP were established by 1H-13C-two-dimensional NMR. Both are competitive inhibitors of the cytochrome bc 1 complex, with TMDMP being the stronger one. Preliminary results suggest that TMDMP binds tightly enough to make X-ray crystallography of inhibitor–bc 1 complex co-crystals feasible. The binding site of TMDMP does not overlap with the binding sites of stigmatellin, MOA-stilbene (MOAS), undecylhydroxydioxobenzothiazole (UHDBT) and myxothaizol.
Keywords: Nonoxidizable; Ubiquinol; Cytochrome bc 1 complex;

Mitochondrial damage by nitric oxide is potentiated by dopamine in PC12 cells by Fernando Antunes; Derick Han; Daniel Rettori; Enrique Cadenas (233-238).
Mitochondrial damage in PC12 cells, a model for dopaminergic cells, was examined in terms of the contribution of oxidative stress, nitric oxide (NO), and dopamine to impairment of mitochondrial respiratory control (RC). A kinetic analysis suggested that the oxidative deamination of dopamine catalyzed by monoamine oxidase (MAO) was not a significant source of hydrogen peroxide, because of constrains imposed by the low cytosolic level of dopamine. NO induced irreversible damage of mitochondrial complex I in PC12 cells: this damage followed a sigmoid response on NO concentration with a well-defined threshold level. Dopamine did not elicit damage of mitochondria in PC12 cells; however, the amine potentiated the effects of NO at or near the threshold level, thus leading to irreversible impairment of mitochondrial respiration. This synergism between NO and dopamine was not observed at NO concentrations below the threshold level. Depletion of dopamine from the storage vesicles by reserpine protected mitochondria from NO damage. Dopamine oxidation by NO increased with pH, and occurred at modest levels at pH 5.5. In spite of this, calculations showed that the oxidation of dopamine in the storage vesicles (pH 5.5) was higher than that in the cytosol (pH 7.4), due to the higher dopamine concentration in the storage vesicles (millimolar range) compared to that in the cytosol (micromolar range). It is suggested that storage vesicles may be the cellular sites where the potential for dopamine oxidation by NO is higher.These data provide further support to the hypothesis that dopamine renders dopaminergic cells more susceptible to the mitochondrial damaging effects of NO. In the early stages of Parkinson's disease, NO production increases until reaching a point near the threshold level that induces neuronal damage. Dopamine stored in dopaminergic cells may cause these cells to be more susceptible to the deleterious effects of NO, which involve irreversible impairment of mitochondrial respiration.
Keywords: Nitric oxide; Mitochondrion; Storage vesicle; Complex I; Reserpine; Synergism;

Green leaves illuminated with photosynthetically active light emit red fluorescence, whose time-dependent intensity variations reflect photosynthetic electron transport (the Kautsky effect). Usually, fluorescence variations are discussed by considering only the contribution of PSII-associated chlorophyll a, although it is known that the fluorescence of PSI-associated chlorophyll a also contributes to the total fluorescence [Aust. J. Plant Physiol. 22 (1995) 131]. Because the fluorescence emitted by each photosystem cannot be measured separately by selecting the emission wavelength in in vivo conditions, the contribution of PSI to total fluorescence at room temperature is still in ambiguity. By using a diode array detector, we measured fluorescence emission spectra corresponding to the minimal (F O) and maximal (F M) fluorescence states. We showed that the different shapes of these spectra were mainly due to a higher contribution of PSI chlorophylls in the F O spectrum. By exciting PSI preferentially, we recorded a reference PSI emission spectrum in the near far-red region. From the F O and F M spectra and from this PSI reference spectrum, we derived specific PSI and PSII emission spectra in both the F O and F M states. This enables to estimate true value of the relative variable fluorescence of PSII, which was underestimated in previous works. Accurate separation of PSI–PSII fluorescence emission spectra will also enable further investigations of the distribution of excitation energy between PSI and PSII under in vivo conditions.
Keywords: Chlorophyll fluorescence; Energy distribution; PSI; PSII;

The phospholipid composition of Rhodopseudomonas acidophila strain 10050 grown aerobically or anaerobically in the light was determined. The major phospholipids present in the aerobic cells were phosphatidylethanolamine (PE; 54%), phosphatidylglycerol (PG; 24%) and cardiolipin (diphosphatidylglycerol, DPG) (14%), together with phosphatidylcholine (PC; 5%). On moving the cells to anaerobic photosynthetic growth in the light PE remained the major phospholipid (37–49%), but there was a major change in the proportion of PC, which increased to 31–33%, and corresponding reductions in the contents of PG to 11–16% and DPG to 4–5%. The fatty acid composition of the phospholipids was unusual, compared with other purple non-sulfur photosynthetic bacteria, in that it contained 16:0 (29%), 17:1 (20%) and 19:1 (9%) plus several mainly unsaturated 2-OH fatty acids (9% total) as major components, when grown aerobically in the dark. In contrast when grown photosynthetically under anaerobic conditions there was <2% 17:1 or 19:1 present, while the amounts of 16:1 and 18:1 increased, and 16:0 decreased. The phospholipid composition of the purified light-harvesting complex 2 (LH2) complex was PE (43%), PC (42%) and DPG (15%). Unexpectedly, there was no PG associated with the purified LH2. These findings contrast with previous studies on several other photosynthetic bacteria, which had shown an increase in PG upon photosynthetic growth [Biochem. J. 181 (1979) 339]. The prior hypothesis that phosphatidyglycerol has some specific role to play in the function of light-harvesting complexes cannot be true for Rps. acidophila. It is suggested that specific integral membrane proteins may strongly influence the phospholipid content of the host membranes into which they are inserted.
Keywords: Photosynthetic bacterium; Antenna complex; Purple non-sulfur bacterium; Rhodopseudomonas acidophila; Light harvesting complex; Phospholipid; Fatty acid;

Intra-subunit interactions in the environment of the iron–sulfur cluster FX in Photosystem I (PS I) of Synechocystis sp. PCC 6803 were studied by site-directed and second site suppressor mutations. In subunit PsaB, the cysteine ligand (C565) of FX and a conserved aspartate (D566) adjacent to C565 were modified. The resulting mutants D566E, C556S/D566E, C556H/D566E and C565H/D566E did not assemble PS I in the thylakoids of the cyanobacterium. Yet, this is the first report of cells of the second site-suppressor mutant (D566E/L416P) and of second site-directed mutant (C565S/D566E) in PsaB that could grow autotrophically in light and were found to assemble a stable functional PS I containing all three iron–sulfur centers, FX and FA/B. The newly resolved structure of PS I (PDB 1JB0) was used to interpret the functional interactions among the amino acid residues. It is suggested that the stability of FX is supported by a salt bridge formed between D566, which is adjacent to the cysteine ligand C565 of the iron–sulfur cluster located on loop hi, and R703 located at the start of loop jk. Hydrogen bond between R703 and D571 at the start of loop hi further stabilizes the arginine. Lengthening of the side by 1.2 Å chain in mutation D566E caused destabilization of FX. The extended side-chain was compensated for by the Fe–O, which is 0.3 Å shorter than the Fe–S bond resulting in stabilization of the FX in the double mutations C565S/D566E. The suppressor mutation D566E/L416P allowed greater freedom for the salt bridge E566-R703, thus relieving the pressure introduced by the D566E replacement and enabling the formation of FX. FX and R703 are therefore stabilized through short- and long-range interactions of the inter-helical loops between h–i, j–k and f–g, respectively.
Keywords: Photosystem I; Iron–sulfur cluster; Electron transport; Suppressor mutation; Spectroscopy; Electron paramagnetic resonance;

Spectroscopic properties of PSI–IsiA supercomplexes from the cyanobacterium Synechococcus PCC 7942 by Elena G Andrizhiyevskaya; Tatjana M.E Schwabe; Marta Germano; Sandrine D'Haene; Jochen Kruip; Rienk van Grondelle; Jan P Dekker (265-272).
The cyanobacterium Synechococcus PCC 7942 grown under iron starvation assembles a supercomplex consisting of a trimeric Photosystem I (PSI) complex encircled by a ring of 18 CP43′ or IsiA light-harvesting complexes [Nature 412 (2001) 745]. Here we present a spectroscopic characterization by temperature-dependent absorption and fluorescence spectroscopy, site-selective fluorescence spectroscopy at 5 K, and circular dichroism of isolated PSI–IsiA, PSI and IsiA complexes from this cyanobacterium grown under iron starvation. The results suggest that the IsiA ring increases the absorption cross-section of PSI by about 100%. Each IsiA subunit binds about 16–17 chlorophyll a (Chl a) molecules and serves as an efficient antenna for PSI. Each of the monomers of the trimeric PSI complex contains two red chlorophylls, which presumably give rise to one exciton-coupled dimer and at 5 K absorb and fluoresce at 703 and 713 nm, respectively. The spectral properties of these C-703 chlorophylls are not affected by the presence of the IsiA antenna ring. The spectroscopic properties of the purified IsiA complexes are similar to those of the related CP43 complex from plants, except that the characteristic narrow absorption band of CP43 at 682.5 nm is missing in IsiA.
Keywords: Photosystem I; PSI-IsiA; CP43′; Synechococcus PCC 7942;

Direct electron transfer between hemoglobin and a glassy carbon electrode facilitated by lipid-protected gold nanoparticles by Xiaojun Han; Wenlong Cheng; Zheling Zhang; Shaojun Dong; Erkang Wang (273-277).
We synthesized a kind of gold nanoparticle protected by a synthetic lipid (didodecyldimethylammonium bromide, DDAB). With the help of these gold nanoparticles, hemoglobin can exhibit a direct electron transfer (DET) reaction. The formal potential locates at −169 mV vs. Ag/AgCl. Spectral data indicated the hemoglobin on the electrode was not denatured. The lipid-protected gold nanoparticles were very stable (for at least 8 months). Their average diameter is 6.42 nm. It is the first time to use monolayer-protected nanoparticles to realize the direct electrochemistry of protein.
Keywords: Hemoglobin; Lipid-protected nanoparticle; Direct electron transfer; UV–Vis spectrum;

Author Index (278-280).

Cumulative Contents (281-282).