BBA - Bioenergetics (v.1709, #1)

Photoprotection by carotenoids is generally considered to be based on the photophysical quenching of triplets and singlet oxygen. There is also accumulating evidence of an alternative, chemical quenching of triplets and singlet oxygen by carotenoids. We report the identification of relatively stable cyclic mono- and diendoperoxides as first products of such an alternative reaction. Nevertheless, these species remain reactive and in the dark cause autooxidation of β-carotene in our model system. Their formation could explain the intriguing pro-oxidant and cytotoxic activity of carotenoids.
Keywords: Carotenoid; Chemical quenching of ROS; Cycloaddition; Peroxide; Singlet oxygen;

This review is focused on the mechanism of ubiquinol oxidation by the cytochrome bc 1 complex (bc 1). This integral membrane complex serves as a “hub” in the vast majority of electron transfer chains. The bc 1 oxidizes a ubiquinol molecule to ubiquinone by a unique “bifurcated” reaction where the two released electrons go to different acceptors: one is accepted by the mobile redox active domain of the [2Fe–2S] iron–sulfur Rieske protein (FeS protein) and the other goes to cytochrome b. The nature of intermediates in this reaction remains unclear. It is also debatable how the enzyme prevents short-circuiting that could happen if both electrons escape to the FeS protein. Here, I consider a reaction mechanism that (i) agrees with the available experimental data, (ii) entails three traits preventing the short-circuiting in bc 1, and (iii) exploits the evident structural similarity of the ubiquinone binding sites in the bc 1 and the bacterial photosynthetic reaction center (RC). Based on the latter congruence, it is suggested that the reaction route of ubiquinol oxidation by bc 1 is a reversal of that leading to the ubiquinol formation in the RC. The rate-limiting step of ubiquinol oxidation is then the re-location of a ubiquinol molecule from its stand-by site within cytochrome b into a catalytic site, which is formed only transiently, after docking of the mobile redox domain of the FeS protein to cytochrome b. In the catalytic site, the quinone ring is stabilized by Glu-272 of cytochrome b and His-161 of the FeS protein. The short circuiting is prevented as long as: (i) the formed semiquinone anion remains bound to the reduced FeS domain and impedes its undocking, so that the second electron is forced to go to cytochrome b; (ii) even after ubiquinol is fully oxidized, the reduced FeS domain remains docked to cytochrome b until electron(s) pass through cytochrome b; (iii) if cytochrome b becomes (over)reduced, the binding and oxidation of further ubiquinol molecules is hampered; the reason is that the Glu-272 residue is turned towards the reduced hemes of cytochrome b and is protonated to stabilize the surplus negative charge; in this state, this residue cannot participate in the binding/stabilization of a ubiquinol molecule.
Keywords: Electron transfer; Proton transfer; Ubiquinone; Photosynthetic reaction center; Membrane potential; Protonmotive force; Rhodobacter sphaeroides; Rhodobacter capsulatus;

The mitochondrial uncoupling proteins UCP2 and UCP3 may be important in attenuating mitochondrial production of reactive oxygen species, in insulin signalling (UCP2), and perhaps in thermogenesis and other processes. To understand their physiological roles, it is necessary to know what reactions they are able to catalyse. We critically examine the evidence for proton transport and anion transport by UCP2 and UCP3. There is good evidence that they increase mitochondrial proton conductance when activated by superoxide, reactive oxygen species derivatives such as hydroxynonenal, and other alkenals or their analogues. However, they do not catalyse proton leak in the absence of such acute activation. They can also catalyse export of fatty acid and other anions, although the relationship of anion transport to proton transport remains controversial.
Keywords: Uncoupling protein; Mild uncoupling; Superoxide; Reactive oxygen species; Reactive alkenals; Hydroxynonenal;

Investigations into high light and oxidative stress in photosynthetic organisms have focussed primarily on genetic impairment of different photoprotective functions. There are few reports of “gain-of-function” mutations that provide enhanced resistance to high light and/or oxidative stress without reduced productivity. We have isolated at least four such very high light resistant (VHL R ) mutations in the green alga, Chlamydomonas reinhardtii, that permit near maximal growth rates at light intensities lethal to wild type. This resistance is not due to an alteration in electron transport rate or quantity and functionality of the two photosystems that could have enhanced photochemical quenching. Nor is it due to reduced excitation pressure by downregulation of the light harvesting antennae or increased nonphotochemical quenching. In fact, photosynthetic activity is unaffected in more than 30 VHL R isolates. Instead, the basis of the VHL R phenotype is a combination of traits, which appears to be dominated by enhanced capacity to tolerate reactive oxygen species generated by excess light, methylviologen, rose bengal or hydrogen peroxide. This is further evidenced in lower levels of ROS after exposure to very high light in the VHL R -S9 mutant. Additionally, the VHL R phenotype is associated with increased zeaxanthin accumulation, maintenance of fast synthesis and degradation rates of the D1 protein, and sustained balanced electron flow into and out of PSI under very high light. We conclude that the VHL R mutations arose from a selection pressure that favors changes to the regulatory system(s) that coordinates several photoprotective processes amongst which repair of PSII and enhanced detoxification of reactive oxygen species play seminal roles.
Keywords: Chlamydomonas; High light resistance; Photosynthesis; Photosystem I and II; Photoprotection; Photo-oxidative stress;

State transitions in cyanobacteria are a physiological adaptation mechanism that changes the interaction of the phycobilisomes with the Photosystem I and Photosystem II core complexes. A random mutagenesis study in the cyanobacterium Synechocystis sp. PCC6803 identified a gene named rpaC which appeared to be specifically required for state transitions. rpaC is a conserved cyanobacterial gene which was tentatively suggested to code for a novel signal transduction factor. The predicted gene product is a 9-kDa integral membrane protein. We have further examined the role of rpaC by overexpressing the gene in Synechocystis 6803 and by inactivating the ortholog in a second cyanobacterium, Synechococcus sp. PCC7942. Unlike the Synechocystis 6803 null mutant, the Synechococcus 7942 null mutant is unable to segregate, indicating that the gene is essential for cell viability in this cyanobacterium. The Synechocystis 6803 overexpressor is also unable to segregate, indicating that the cells can only tolerate a limited gene copy number. The non-segregated Synechococcus 7942 mutant can perform state transitions but shows a perturbed phycobilisome–Photosystem II interaction. Based on these results, we propose that the rpaC gene product controls the stability of the phycobilisome–Photosystem II supercomplex, and is probably a structural component of the complex.
Keywords: Cyanobacterium; Light-harvesting; Photosynthesis; Photosystem II; Phycobilisome; State transition;

Tobacco rbcL deletion mutant, which lacks the key enzyme Rubisco for photosynthetic carbon assimilation, was characterized with respect to thylakoid functional properties and protein composition. The ΔrbcL plants showed an enhanced capacity for dissipation of light energy by non-photochemical quenching which was accompanied by low photochemical quenching and low overall photosynthetic electron transport rate. Flash-induced fluorescence relaxation and thermoluminescence measurements revealed a slow electron transfer and decreased redox gap between QA and QB, whereas the donor side function of the Photosystem II (PSII) complex was not affected. The 77 K fluorescence emission spectrum of ΔrbcL plant thylakoids implied a presence of free light harvesting complexes. Mutant plants also had a low amount of photooxidisible P700 and an increased ratio of PSII to Photosystem I (PSI). On the other hand, an elevated level of plastid terminal oxidase and the lack of F 0 ‘dark rise’ in fluorescence measurements suggest an enhanced plastid terminal oxidase-mediated electron flow to O2 in ΔrbcL thylakoids. Modified electron transfer routes together with flexible dissipation of excitation energy through PSII probably have a crucial role in protection of PSI from irreversible protein damage in the ΔrbcL mutant under growth conditions. This protective capacity was rapidly exceeded in ΔrbcL mutant when the light level was elevated resulting in severe degradation of PSI complexes.
Keywords: Photosynthesis; Photosystem II; Photosystem I; Photoinhibition; Rubisco;

Role of transmembrane segment 5 of the plant vacuolar H+-pyrophosphatase by Ru C. Van; Yih J. Pan; Shen H. Hsu; Yun T. Huang; Yi Y. Hsiao; Rong L. Pan (84-94).
Vacuolar H+-translocating inorganic pyrophosphatase (V-PPase; EC 3.6.1.1) is a homodimeric proton translocase consisting of a single type of polypeptide with a molecular mass of approximately 81 kDa. Topological analysis tentatively predicts that mung bean V-PPase contains 14 transmembrane domains. Alignment analysis of V-PPase demonstrated that the transmembrane domain 5 (TM5) of the enzyme is highly conserved in plants and located at the N-terminal side of the putative substrate-binding loop. The hydropathic analysis of V-PPase showed a relatively lower degree of hydrophobicity in the TM5 region as compared to other domains. Accordingly, it appears that TM5 is probably involved in the proton translocation of V-PPase. In this study, we used site-directed mutagenesis to examine the functional role of amino acid residues in TM5 of V-PPase. A series of mutants singly replaced by alanine residues along TM5 were constructed and over-expressed in Saccharomyces cerevisiae; they were then used to determine their enzymatic activities and proton translocations. Our results indicate that several mutants displayed minor variations in enzymatic properties, while others including those mutated at E225, a GYG motif (residues from 229 to 231), A238, and R242, showed a serious decline in enzymatic activity, proton translocation, and coupling efficiency of V-PPase. Moreover, the mutation at Y230 relieved several cation effects on the V-PPase. The GYG motif presumably plays a significant role in maintaining structure and function of V-PPase.
Keywords: Proton translocation; Tonoplast; Vacuole; Vacuolar H+-pyrophosphatase; Site-directed mutagenesis; GYG motif;

A nhaD Na + /H + antiporter and a pcd homologues are among the Rhodothermus marinus complex I genes by Ana M.P. Melo; Susana A.L. Lobo; Filipa L. Sousa; Andreia S. Fernandes; Manuela M. Pereira; Gudmundur O. Hreggvidsson; Jacob K. Kristjansson; Lígia M. Saraiva; Miguel Teixeira (95-103).
The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo 1 to nqo 7 (nqoA) and nqo 10 to nqo 14 (nqoB)] and two independent genes (nqo 8 and nqo 9 ). Unexpectedly, two genes encoding homologues of a NhaD Na+/H+ antiporter (NhaD) and of a pterin-4α-carbinolamine dehydratase (PCD) were identified within nqoB, flanked by nqo 13 and nqo 14 . Eight conserved motives to harbour iron–sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqoB. The possible role of these two genes in R. marinus complex I is discussed.
Keywords: Complex I; NADH:quinone oxidoreductase; Rhodothermus marinus; NhaD Na+/H+ antiporter;