BBA - Bioenergetics (v.1777, #6)

Structural stability and properties of three isoforms of the major light-harvesting chlorophyll a/b complexes of photosystem II by Yajie Zhang; Cheng Liu; Shuang Liu; Ye Shen; Tingyun Kuang; Chunhong Yang (479-487).
Three isoforms of the major light-harvesting chlorophyll (Chl) a/b complexs of photosystem II (LHCIIb) in the pea, namely, Lhcb1, Lhcb2, and Lhcb3, were obtained by overexpression of apoprotein in Escherichia coli and by successfully refolding these isoforms with thylakoid pigments in vitro. The sequences of the protein, pigment stoichiometries, spectroscopic characteristics, thermo- and photostabilities of different isoforms were analysed. Comparison of their spectroscopic properties and structural stabilities revealed that Lhcb3 differed strongly from Lhcb1 and Lhcb2 in both respects. It showed the lowest Qy transition energy, with its reddest absorption about 2 nm red-shifted, and the highest photostability under strong illuminations. Among the three isoforms, Lhcb 2 showed lowest thermal stability regarding energy transfer from Chl b to Chl a in the complexes, which implies that the main function of Lhcb 2 under high temperature stress is not the energy transfer.
Keywords: Major light-harvesting chlorophyll a/b complex of photosystem II; Pigment stoichiometry; Thermostability; Photostability; Reconstitution;

Characterization of photosystem II in salt-stressed cyanobacterial Spirulina platensis cells by Hongmei Gong; Yunlai Tang; Jia Wang; Xiaogang Wen; Lixin Zhang; Congming Lu (488-495).
PSII activity was inhibited after Spirulina platensis cells were incubated with different salt concentrations (0–0.8 M NaCl) for 12 h. Flash-induced fluorescence kinetics showed that in the absence of DCMU, the half time of the fast and slow components decreased while that of the middle component increased considerably with increasing salt concentration. In the presence of DCMU, fluorescence relaxation was dominated by a 0.6s component in control cells. After salt stress, this was partially replaced by a faster new component with half time of 20–50 ms. Thermoluminescence measurements revealed that S2QA and S2QB recombinations were shifted to higher temperatures in parallel and the intensities of the thermoluminescence emissions were significantly reduced in salt-stressed cells. The period-four oscillation of the thermoluminescence B band was highly damped. There were no significant changes in contents of CP47, CP43, cytochrome c550, and D1 proteins. However, content of the PsbO protein in thylakoid fraction decreased but increased significantly in soluble fraction. The results suggest that salt stress leads to a modification of the QB niche at the acceptor side and an increase in the stability of the S2 state at the donor side, which is associated with a dissociation of the PsbO protein.
Keywords: Chlorophyll fluorescence; Photosystem II; PsbO protein; Salt stress; Spirulina platensis; Thermoluminescence;

Direct quantification of the four individual S states in Photosystem II using EPR spectroscopy by Guangye Han; Felix M. Ho; Kajsa G.V. Havelius; Susan F. Morvaridi; Fikret Mamedov; Stenbjörn Styring (496-503).
EPR spectroscopy is very useful in studies of the oxygen evolving cycle in Photosystem II and EPR signals from the CaMn4 cluster are known in all S states except S4. Many signals are insufficiently understood and the S0, S1, and S3 states have not yet been quantifiable through their EPR signals. Recently, split EPR signals, induced by illumination at liquid helium temperatures, have been reported in the S0, S1, and S3 states. These split signals provide new spectral probes to the S state chemistry. We have studied the flash power dependence of the S state turnover in Photosystem II membranes by monitoring the split S0, split S1, split S3 and S2 state multiline EPR signals. We demonstrate that quantification of the S1, S3 and S0 states, using the split EPR signals, is indeed possible in samples with mixed S state composition. The amplitudes of all three split EPR signals are linearly correlated to the concentration of the respective S state. We also show that the S1 → S2 transition proceeds without misses following a saturating flash at 1 °C, whilst substantial misses occur in the S2 → S3 transition following the second flash.
Keywords: Photosystem II; Oxygen evolving complex; S states; Split signals; EPR; Misses;

Proteorhodopsin is photoactive 7-transmembrane protein, which uses all-trans retinal as a chromophore. Proteorhodopsin subfamilies are spectrally tuned in accordance with the depth of habitat of the host organisms, numerous species of marine picoplankton. We try to find residues critical for the spectral tuning through the use of random PCR mutagenesis and endogenous retinal biosynthesis. We obtained 16 isolates with changed color by screening in Escherichia coli with internal retinal biosynthesis system containing genes for beta-carotene biosynthesis and retinal synthase. Some isolates contained multiple substitutions, which could be separated to give 20 single mutations influencing the spectral properties. The color-changing residues are distributed through the protein except for the helix A, and about a half of the mutations is localized on the helices C and D, implying their importance for color tuning. In the pumping form of the pigment, absorption maxima in 8 mutants are red-shifted and in 12 mutants are blue-shifted compared to the wild-type. The results of flash-photolysis showed that most of the low pumping activity mutants possess slower rates of M decay and O decay. These results suggest that the color-tuning residues are not restricted to the retinal binding pocket, in accord with a recent evolutionary analysis.
Keywords: Proteorhodopsin; Spectral tuning; Membrane protein; Proton pumping; Random mutagenesis;

Distinct organization of energy metabolism in HL-1 cardiac cell line and cardiomyocytes by Margus Eimre; Kalju Paju; Sophie Pelloux; Nathalie Beraud; Mart Roosimaa; Lumme Kadaja; Marju Gruno; Nadezhda Peet; Ehte Orlova; Reele Remmelkoor; Andres Piirsoo; Valdur Saks; Enn Seppet (514-524).
Expression and function of creatine kinase (CK), adenylate kinase (AK) and hexokinase (HK) isoforms in relation to their roles in regulation of oxidative phosphorylation (OXPHOS) and intracellular energy transfer were assessed in beating (B) and non-beating (NB) cardiac HL-l cell lines and adult rat cardiomyocytes or myocardium. In both types of HL-1 cells, the AK2, CKB, HK1 and HK2 genes were expressed at higher levels than the CKM, CKMT2 and AK1 genes. Contrary to the saponin-permeabilized cardiomyocytes the OXPHOS was coupled to mitochondrial AK and HK but not to mitochondrial CK, and neither direct transfer of adenine nucleotides between CaMgATPases and mitochondria nor functional coupling between CK-MM and CaMgATPases was observed in permeabilized HL-1 cells. The HL-1 cells also exhibited deficient complex I of the respiratory chain. In conclusion, contrary to cardiomyocytes where mitochondria and CaMgATPases are organized into tight complexes which ensure effective energy transfer and feedback signaling between these structures via specialized pathways mediated by CK and AK isoforms and direct adenine nucleotide channeling, these complexes do not exist in HL-1 cells due to less organized energy metabolism.
Keywords: HL-1 cell; Heart; Energy transfer; Kinase; Gene expression;

A role for redox-active tyrosines has been demonstrated in many important biological processes, including water oxidation carried out by photosystem II (PSII) of oxygenic photosynthesis. The rates of tyrosine oxidation and reduction and the Tyr/Tyr reduction potential are undoubtedly controlled by the immediate environment of the tyrosine, with the coupling of electron and proton transfer, a critical component of the kinetic and redox behavior. It has been demonstrated by Faller et al. that the rate of oxidation of tyrosine D (TyrD) at room temperature and the extent of TyrD oxidation at cryogenic temperatures, following flash excitation, dramatically increase as a function of pH with a pK a of ≈ 7.6 [Faller et al. 2001 Proc. Natl. Acad. Sci. USA 98, 14368–14373; Faller et al. 2001 Biochemistry 41, 12914–12920]. In this work, we investigated, using FTIR difference spectroscopy, the mechanistic reasons behind this large pH dependence. These studies were carried out on Mn-depleted PSII core complexes isolated from Synechocystis sp. PCC 6803, WT unlabeled and labeled with 13C6-, or 13C1(4)-labeled tyrosine, as well as on the D2-Gln164Glu mutant. The main conclusions of this work are that the pH-induced changes involve the reduced TyrD state and not the oxidized TyrD state and that TyrD does not exist in the tyrosinate form between pH 6 and 10. We can also exclude a change in the protonation state of D2-His189 as being responsible for the large pH dependence of TyrD oxidation. Indeed, our data are consistent with D2-His189 being neutral both in the TyrD and TyrD states in the whole pH6-10 range. We show that the interactions between reduced TyrD and D2-His189 are modulated by the pH. At pH greater than 7.5, the ν(CO) mode frequency of TyrD indicates that TyrD is involved in a strong hydrogen bond, as a hydrogen bond donor only, in a fraction of the PSII centers. At pH below 7.5, the hydrogen-bonding interaction formed by TyrD is weaker and TyrD could be also involved as a hydrogen bond acceptor, according to calculations performed by Takahashi and Noguchi [J. Phys. Chem. B 2007 111, 13833–13844]. The involvement of TyrD in this strong hydrogen-bonding interaction correlates with the ability to oxidize TyrD at cryogenic temperatures and rapidly at room temperature. A strong hydrogen-bonding interaction is also observed at pH 6 in the D2-Gln164Glu mutant, showing that the residue at position D2-164 regulates the properties of TyrD. The IR data point to the role of a protonatable group(s) (with a pK a of ≈ 7) other than D2-His189 and TyrD, in modifying the characteristics of the TyrD hydrogen-bonding interactions, and hence its oxidation properties. It remains to be determined whether the strong hydrogen-bonding interaction involves D2-His189 and if TyrD oxidation involves the same proton transfer route at low and at high pH.
Keywords: FTIR; Photosystem II; Redox-active tyrosine; Synechocystis PCC 6803; Tyrosyl radical; TyrD;

Hydrogencarbonate is not a tightly bound constituent of the water-oxidizing complex in photosystem II by Dmitriy Shevela; Ji-Hu Su; Vyacheslav Klimov; Johannes Messinger (532-539).
Since the end of the 1950s hydrogencarbonate (‘bicarbonate’) is discussed as a possible cofactor of photosynthetic water-splitting, and in a recent X-ray crystallography model of photosystem II (PSII) it was displayed as a ligand of the Mn4O x Ca cluster. Employing membrane-inlet mass spectrometry (MIMS) and isotope labelling we confirm the release of less than one (≈ 0.3) HCO3 per PSII upon addition of formate. The same amount of HCO3 release is observed upon formate addition to Mn-depleted PSII samples. This suggests that formate does not replace HCO3 from the donor side, but only from the non-heme iron at the acceptor side of PSII. The absence of a firmly bound HCO3 is corroborated by showing that a reductive destruction of the Mn4O x Ca cluster inside the MIMS cell by NH2OH addition does not lead to any CO2/HCO3 release. We note that even after an essentially complete HCO3 /CO2 removal from the sample medium by extensive degassing in the MIMS cell the PSII samples retain ≥ 75% of their initial flash-induced O2-evolving capacity. We therefore conclude that HCO3 has only ‘indirect’ effects on water-splitting in PSII, possibly by being part of a proton relay network and/or by participating in assembly and stabilization of the water-oxidizing complex.
Keywords: Membrane-inlet mass spectrometry (MIMS); Photosystem II; Water-splitting; Water oxidation; Hydrogencarbonate; Bicarbonate;

Both ATP-regulated (mitoKATP) and large conductance calcium-activated (mitoBKCa) potassium channels have been proposed to regulate mitochondrial K+ influx and matrix volume and to mediate cardiac ischaemic preconditioning (IP). However, the specificity of the pharmacological agents used in these studies and the mechanisms underlying their effects on IP remain controversial. Here we used increasing concentrations of K+-ionophore (valinomycin) to stimulate respiration by rat liver and heart mitochondria in the presence of the K+/H+ exchanger nigericin. This allowed rates of valinomycin-induced K+ influx to be determined whilst parallel measurements of light scattering (A520) and matrix volume (3H2O and [14C]-sucrose) enabled rates of K+ influx to be correlated with increases in matrix volume. Light scattering readily detected an increase in K+ influx of < 5 nmol K+ min− 1 per mg protein corresponding to < 2% mitochondrial matrix volume increase. In agreement with earlier data no light-scattering changes were observed in response to any mitoKATP channel openers or blockers. However, the mitoBKCa opener NS1619 (10–50 µM) did decrease light scattering slightly, but this was also seen in K+-free medium and was accompanied by uncoupling. Contrary to prediction, the mitoBKCa blocker paxilline (10–50 µM) decreased rather than increased light scattering, and it also slightly uncoupled respiration. Our data argue against the presence of significant activities of either the mitoKATP or the mitoBKCa channel in rat liver and heart mitochondria and provide further evidence that preconditioning induced by pharmacological openers of these channels is more likely to involve alternative mechanisms.
Keywords: Mitochondria; Mitochondrial ATP-regulated potassium channel; Mitochondrial large conductance calcium-activated potassium channel; Mitochondrial volume; Ischaemic preconditioning;