BBA - Bioenergetics (v.1777, #12)

The mitochondrial paradigm for a chemiosmotic energy transduction mechanism requires frequently misunderstood modifications for application to microbes growing and surviving at acidic pH values and/or with relatively weak reductants as energy sources. Here the bioenergetics of the iron oxidiser Acidithiobacillus ferrooxidans are reviewed and analysed so as to develop the general bioenergetic principles for understanding organisms that grow under these conditions. Extension of the principles outlined herein to organisms that survive (as opposed to grow) under these conditions is to be presented in a subsequent article.
Keywords: Acidithiobacillus; Inverse membrane potential; Reversed electron transport; Cytochrome oxidase;

The organisation of proton motive and non-proton motive redox loops in prokaryotic respiratory systems by Jörg Simon; Rob J.M. van Spanning; David J. Richardson (1480-1490).
Respiration is fundamental to the aerobic and anaerobic energy metabolism of many prokaryotic and most eukaryotic organisms. In principle, the free energy of a redox reaction catalysed by a membrane-bound electron transport chain is transduced via the generation of an electrochemical ion (usually proton) gradient across a coupling membrane that drives ATP synthesis. The proton motive force (pmf) can be built up by different mechanisms like proton pumping, quinone/quinol cycling or by a redox loop. The latter couples electron transport to a net proton transfer across the membrane without proton pumping. Instead, charge separation is achieved by quinone-reactive enzymes or enzyme complexes whose active sites for substrates and quinones are situated on different sides of the coupling membrane. The necessary transmembrane electron transport is usually accomplished by the presence of two haem groups that face opposite sides of the membrane. There are many different enzyme complexes that are part of redox loops and their catalysed redox reactions can be either electrogenic, electroneutral (non-proton motive) or even pmf-consuming. This article gives conceptual classification of different operational organisations of redox loops and uses this as a platform from which to explore the biodiversity of quinone/quinol-cycling redox systems.
Keywords: Aerobic and anaerobic respiration; Proton motive force; Redox (half) loop; Electron transport chain; Quinone reductase; Quinol dehydrogenase;

Understanding chlorophylls: Central magnesium ion and phytyl as structural determinants by Leszek Fiedor; Agnieszka Kania; Beata Myśliwa-Kurdziel; Łukasz Orzeł; Grażyna Stochel (1491-1500).
Phytol, a C20 alcohol esterifying the C-173 propionate, and Mg2+ ion chelated in the central cavity, are conservative structural constituents of chlorophylls. To evaluate their intramolecular structural effects we prepared a series of metal- and phytyl-free derivatives of bacteriochlorophyll a and applied them as model chlorophylls. A detailed spectroscopic study on the model pigments reveals meaningful differences in the spectral characteristics of the phytylated and non-phytylated pigments. Their analysis in terms of solvatochromism and axial coordination shows how the central Mg and phytyl residue shape the properties of the pigment. Surprisingly, the presence/absence of the central Mg has no effect on the solvatochromism of (bacterio)chlorophyll π-electron system and the hydrophobicity of phytyl does not interfere with the first solvation shell of the chromophore. However, both residues significantly influence the conformation of the pigment macrocycle and the removal of either residue increases the macrocycle flexibility. The chelation of Mg has a flattening effect on the macrocycle whereas bulky phytyl residue seems to control the conformation of the chromophore via steric interactions with ring V and its substituents. The analysis of spectroscopic properties of bacteriochlorophyllide (free acid) shows that esterification of the C-173 propionate is necessary in chlorophylls because the carboxyl group may act as a strong chelator of the central Mg. These observations imply that the truncated chlorophylls used in theoretical studies are not adequate as models of native chromophores, especially when fine effects are to be modeled.
Keywords: Bacteriochlorophyll; Phytol; Magnesium ion; Solvatochromism; Axial coordination; Macrocycle planarity;

Isolation of highly active photosystem II core complexes with a His-tagged Cyt b 559 subunit from transplastomic tobacco plants by Holger Fey; Dario Piano; Ruth Horn; David Fischer; Matthias Schmidt; Stephanie Ruf; Wolfgang P. Schröder; Ralph Bock; Claudia Büchel (1501-1509).
Photosystem II (PSII) is a huge multi-protein-complex consisting, in higher plants and green algae, of the PS II core and the adjacent light harvesting proteins. In the study reported here, N-terminal His-tags were added to the plastome-encoded α-subunit of cytochrome b 559 , PsbE, in tobacco plants, thus facilitating rapid, mild purification of higher plant PSII. Biolistic chloroplast transformation was used to replace the wildtype psbE gene by His-tagged counterparts. Transgenic plants did not exhibit an obvious phenotype. However, the oxygen evolution capacity of thylakoids prepared from the mutants compared to the wildtype was reduced by 10–30% depending on the length of the His-tag, although Fv/Fm values differed only slightly. Homoplasmic F1 plants were used to isolate PSII cores complexes. The cores contained no detectable traces of LHC or PsaA/B polypeptides, but the main core subunits of PSII could be identified using immunodetection and mass spectroscopy. In addition, Psb27 and PsbS were detected. The presence of the former was presumably due to the preparation method, since PSII complexes located in the stroma are also isolated. In contrast to previous reports, PsbS was solely found as a monomer on SDS-PAGE in the PSII core complexes of tobacco.
Keywords: PsbE; Psb27; PsbS; Non-photochemical quenching;

The role of TyrD in the electron transfer kinetics in Photosystem II by Malwina Szczepaniak; Miwa Sugiura; Alfred R. Holzwarth (1510-1517).
Redox-active tyrosine (Tyr) D is indirectly involved in controlling the primary electron transfer in PSII. The presence of the oxidized TyrD renders P680+ more oxidizing by localizing the charge more on PD1 and thus facilitates trapping of the excitation energy in PSII. We also conclude that the mechanism of the primary charge separation and stabilization is altered upon QA reduction.
Keywords: Photosystem II; Electron transfer; Fluorescence kinetics; Charge separation; Tyrosine D; Photosynthesis; Picosecond kinetics; Ultrafast spectroscopy;

Intrinsic uncoupling in the ATP synthase of Escherichia coli by Manuela D'Alessandro; Paola Turina; B. Andrea Melandri (1518-1527).
The ATP hydrolysis activity and proton pumping of the ATP synthase of Escherichia coli in isolated native membranes have been measured and compared as a function of ADP and Pi concentration. The ATP hydrolysis activity was inhibited by Pi with an half-maximal effect at 140 μM, which increased progressively up in the millimolar range when the ADP concentration was progressively decreased by increasing amounts of an ADP trap. In addition, the relative extent of this inhibition decreased with decreasing ADP. The half-maximal inhibition by ADP was found in the submicromolar range, and the extent of inhibition was enhanced by the presence of Pi. The parallel measurement of ATP hydrolysis activity and proton pumping indicated that, while the rate of ATP hydrolysis was decreased as a function of either ligand, the rate of proton pumping increased. The latter showed a biphasic response to the concentration of Pi, in which an inhibition followed the initial stimulation. Similarly as previously found for the ATP synthase from Rhodobacter caspulatus [P. Turina, D. Giovannini, F. Gubellini, B.A. Melandri, Physiological ligands ADP and Pi modulate the degree of intrinsic coupling in the ATP synthase of the photosynthetic bacterium Rhodobacter capsulatus, Biochemistry 43 (2004) 11126–11134], these data indicate that the E. coli ATP synthase can operate at different degrees of energetic coupling between hydrolysis and proton transport, which are modulated by ADP and Pi.
Keywords: E. coli; ATP synthase; Uncoupling; H+/ATP; Stoichiometry; Pi;

The haem–copper oxygen reductase of Desulfovibrio vulgaris contains a dihaem cytochrome c in subunit II by Susana A.L. Lobo; Claúdia C. Almeida; João N. Carita; Miguel Teixeira; Lígia M. Saraiva (1528-1534).
The genome of the sulphate reducing bacterium Desulfovibrio vulgaris Hildenborough, still considered a strict anaerobe, encodes two oxygen reductases of the bd and haem–copper types. The haem–copper oxygen reductase deduced amino acid sequence reveals that it is a Type A2 enzyme, which in its subunit II contains two c-type haem binding motifs. We have characterized the cytochrome c domain of subunit II and confirmed the binding of two haem groups, both with Met-His iron coordination. Hence, this enzyme constitutes the first example of a ccaa 3 haem–copper oxygen reductase. The expression of D. vulgaris haem–copper oxygen reductase was found to be independent of the electron donor and acceptor source and is not altered by stress factors such as oxygen exposure, nitrite, nitrate, and iron; therefore the haem–copper oxygen reductase seems to be constitutive. The KCN sensitive oxygen reduction by D. vulgaris membranes demonstrated in this work indicates the presence of an active haem–copper oxygen reductase. D. vulgaris membranes perform oxygen reduction when accepting electrons from the monohaem cytochrome c 553, thus revealing the first possible electron donor to the terminal oxygen reductase of D. vulgaris. The physiological implication of the presence of the oxygen reductase in this organism is discussed.
Keywords: Haem–copper oxygen reductase; Desulfovibrio; Oxygen reduction; Cytochrome c 553;

The photosynthetic reaction center from the green sulfur bacterium Chlorobium tepidum (CbRC) was solubilized from membranes using Triton X-100 and isolated by sucrose density ultra-centrifugation. The CbRC complexes were subsequently treated with 0.5 M NaCl and ultrafiltered over a 100 kDa cutoff membrane. The resulting CbRC cores did not exhibit the low-temperature EPR resonances from FA and FB and were unable to reduce NADP+. SDS-PAGE and mass spectrometric analysis showed that the PscB subunit, which harbors the FA and FB clusters, had become dissociated, and was now present in the filtrate. Attempts to rebind PscB onto CbRC cores were unsuccessful. Mössbauer spectroscopy showed that recombinant PscB contains a heterogeneous mixture of [4Fe–4S]2+,1+ and other types of Fe/S clusters tentatively identified as [2Fe–2S]2+,1+ clusters and rubredoxin-like Fe3+,2+ centers, and that the [4Fe–4S]2+,1+ clusters which were present were degraded at high ionic strength. Quantitative analysis confirmed that the amount of iron and sulfide in the recombinant protein was sub-stoichiometric. A heme-staining assay indicated that cytochrome c 551 remained firmly attached to the CbRC cores. Low-temperature EPR spectroscopy of photoaccumulated CbRC complexes and CbRC cores showed resonances between g  = 5.4 and 4.4 assigned to a S  = 3/2 ground spin state [4Fe–4S]1+ cluster and at g  = 1.77 assigned to a S  = 1/2 ground spin state [4Fe–4S]1+ cluster, both from FX . These results unify the properties of the acceptor side of the Type I homodimeric reaction centers found in green sulfur bacteria and heliobacteria: in both, the FA and FB iron–sulfur clusters are present on a salt-dissociable subunit, and FX is present as an interpolypeptide [4Fe–4S]2+,1+ cluster with a significant population in a S  = 3/2 ground spin state.
Keywords: Chlorobium; EPR; Iron–sulfur cluster; FMO protein;

A novel protein in Photosystem II of a diatom Chaetoceros gracilis is one of the extrinsic proteins located on lumenal side and directly associates with PSII core components by Akinori Okumura; Ryo Nagao; Takehiro Suzuki; Satoshi Yamagoe; Masako Iwai; Katsuyoshi Nakazato; Isao Enami (1545-1551).
The gene encoding a novel extrinsic protein (Psb31) found in Photosystem II (PSII) of a diatom, Chaetoceros gracilis, was cloned and sequenced. The deduced protein contained three characteristic leader sequences targeted for chloroplast endoplasmic reticulum membrane, chloroplast envelope membrane and thylakoid membrane, indicating that Psb31 is encoded in the nuclear genome and constitutes one of the extrinsic proteins located on the lumenal side. Homologous genes were found in a red alga and chromophytic algae but not in other organisms. Genes encoding the other four extrinsic proteins in C. gracilis PSII were also cloned and sequenced, and their leader sequences were characterized and compared. To search for the nearest neighbor relationship between Psb31 and the other PSII components, we crosslinked the PSII particles with the water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, and found that Psb31 directly associates with PSII core components through electrostatic interaction, suggesting that the novel Psb31 protein is one of the extrinsic proteins constituting the functional oxygen-evolving complex of C. gracilis PSII.
Keywords: Diatom; Extrinsic protein; Oxygen evolution; Photosystem II; Psb31; Chaetoceros gracilis;

Organisation and function of the Phaeospirillum molischianum photosynthetic apparatus by Camille Mascle-Allemand; Jérôme Lavergne; Alain Bernadac; James N. Sturgis (1552-1559).
We have investigated the organisation of the photosynthetic apparatus in Phaeospirillum molischianum, using biochemical fractionation and functional kinetic measurements. We show that only a fraction of the ATP-synthase is present in the membrane regions which contain most of the photosynthetic apparatus and that, despite its complicated stacked structure, the intracytoplasmic membrane delimits a single connected space. We find that the diffusion time required for a quinol released by the reaction centre to reach a cytochrome bc 1 complex is about 260 ms. On the other hand, the reduction of the cytochrome c chain by the cytochrome bc 1 complex in the presence of a reduced quinone pool occurs with a time constant of about 5 ms. The overall turnover time of the cyclic electron transfer is about 25 ms in vivo under steady-state illumination. The sluggishness of the quinone shuttle appears to be compensated, at least in part, by the size of the quinone pool. Together, our results show that P. molischianum contains a photosynthetic system, with a very different organisation from that found in Rhodobacter sphaeroides, in which quinone/quinol diffusion between the RC and the cytochrome bc 1 is likely to be the rate-limiting factor for cyclic electron transfer.
Keywords: Purple bacteria; Photosynthesis; Electron transfer; Light harvesting; Cytochrome; Rhodospirillum molischianum;