BBA - Bioenergetics (v.1757, #7)

In the course of the synthesis of γ-pyrones, well-known inhibitors of photosystem II electron transport, it turned out that the starting material, acyl derivatives of 2,2-dimethyl-1,3-dioxane-5,6-dione (Meldrum's acid) are potent inhibitors of photosystem II electron transport. Thus, in a simple one-step synthesis from commercial available substances, highly potent photosystem II inhibitors are generated. The biological activity of the acyl derivatives is in a parabolic fashion dependent from the length of the alkyl side chain.
Keywords: 2,2-dimethyl-1,3-dioxane-4,6-dione; pyrandione; γ-pyrone; photosystem II; inhibitors;

The emergence of Arabidopsis as a model plant provides an opportunity to gain insights into the role of the alternative oxidase that cannot be as readily achieved in other plant species. The analysis of extensive mRNA expression data indicates that all five Aox genes (Aox1a, 1b, 1c, 1d and 2) are expressed, but organ and developmental regulation are evident, suggesting regulatory specialisation of Aox gene members. The stress-induced nature of the alternative pathway in a variety of plants is further supported in Arabidopsis as Aox1a and Aox1d are amongst the most stress responsive genes amongst the hundreds of known genes encoding mitochondrial proteins. Analysis of genes co-expressed with Aoxs from studies of responses to various treatments altering mitochondrial functions and/or from plants with altered Aox levels reveals that: (i) this gene set encodes more functions outside the mitochondrion than functions in mitochondria, (ii) several pathways for induction exist and there is a difference in the magnitude of the induction in each pathway, (iii) the magnitude of induction may depend on the endogenous levels of Aox, and (iv) induction of Aox can be oxidative stress-dependent or -independent depending on the gene member and the tissue analysed. An overall role for Aox in re-programming cellular metabolism in response to the ever changing environment encountered by plants is proposed.
Keywords: Alternative oxidase; Arabidopsis thaliana; Multi-gene family; Co-expression; Function; Stress;

A new paradigm for the action of reactive oxygen species in the photoinhibition of photosystem II by Yoshitaka Nishiyama; Suleyman I. Allakhverdiev; Norio Murata (742-749).
Inhibition of the activity of photosystem II (PSII) under strong light is referred to as photoinhibition. This phenomenon is due to the imbalance between the rate of photodamage to PSII and the rate of the repair of damaged PSII. Photodamage is initiated by the direct effects of light on the oxygen-evolving complex and, thus, photodamage to PSII is unavoidable. Studies of the effects of oxidative stress on photodamage and subsequent repair have revealed that reactive oxygen species (ROS) act primarily by inhibiting the repair of photodamaged PSII and not by damaging PSII directly. Thus, strong light has two distinct effects on PSII; it damages PSII directly and it inhibits the repair of PSII via production of ROS. Investigations of the ROS-induced inhibition of repair have demonstrated that ROS suppress the synthesis de novo of proteins and, in particular, of the D1 protein, that are required for the repair of PSII. Moreover, a primary target for inhibition by ROS appears to be the elongation step of translation. Inhibition of the repair of PSII by ROS is accelerated by the deceleration of the Calvin cycle that occurs when the availability of CO2 is limited. In this review, we present a new paradigm for the action of ROS in photoinhibition.
Keywords: Photoinhibition; Photosystem II; Protein synthesis; Reactive oxygen species; Repair;

Myoglobin with modified tetrapyrrole chromophores: Binding specificity and photochemistry by Stephanie Pröll; Brigitte Wilhelm; Bruno Robert; Hugo Scheer (750-763).
Complexes were prepared of horse heart myoglobin with derivatives of (bacterio)chlorophylls and the linear tetrapyrrole, phycocyanobilin. Structural factors important for binding are (i) the presence of a central metal with open ligation site, which even induces binding of phycocyanobilin, and (ii) the absence of the hydrophobic esterifying alcohol, phytol. Binding is further modulated by the stereochemistry at the isocyclic ring. The binding pocket can act as a reaction chamber: with enolizable substrates, apo-myoglobin acts as a 132-epimerase converting, e.g., Zn-pheophorbide a' (132S) to a (132R). Light-induced reduction and oxidation of the bound pigments are accelerated as compared to solution. Some flexibility of the myoglobin is required for these reactions to occur; a nucleophile is required near the chromophores for photoreduction (Krasnovskii reaction), and oxygen for photooxidation. Oxidation of the bacteriochlorin in the complex and in aqueous solution continues in the dark.
Keywords: Chlorophyll, transmetalated; Myoglobin binding site; Photochemistry; Stereochemistry, photosynthesis; Photodynamic therapy;

In mammalian cardiomyocytes, mitochondria and adjacent ATPases with participation of creatine kinase (CK) constitute functional compartments with an exchange of ADP and ATP delimited from cytosolic bulk solution. The question arises if this extends to ectothermic vertebrates: their low body temperature and thinner cardiomyocytes with a lower density of membrane structures may reduce the need and structural basis for compartmentation. In saponin-skinned cardiac fibres from rainbow trout and Atlantic cod, we investigated mitochondrial respiration induced by endogenous ADP generated by ATPases and its competition for this ADP with pyruvate kinase (PK) in excess. At low Ca2+ activity (pCa = 7.0), PK lowered ATP-induced respiration by 40% in trout and 26% in cod. At high Ca2+ activity (pCa = 5.41), PK had no effect. Additionally, ADP release from the fibres was almost zero but increased drastically upon inhibition of respiration with 1 mM Na-azide. This suggests that fibres are compartmented. PK abolished creatine-stimulated respiration in trout suggesting a less tight coupling of CK to respiration than in mammals. In conclusion, intracellular compartmentation seems to be a general feature of vertebrate cardiomyocytes, whereas the role of CK is unclear, but it seems to be less important for energy transport in species with lower metabolism.
Keywords: Ectothermic vertebrate; Rainbow trout; Atlantic cod; Cardiomyocyte; Mitochondrial respiration; Intracellular compartmentation;

Opposite effects of d-fructose on total versus cytosolic ATP/ADP ratio in pancreatic islet cells by Marie-Hélène Giroix; Eda Agascioglu; Berrin Oguzhan; Karim Louchami; Ying Zhang; Philippe Courtois; Willy J. Malaisse; Abdullah Sener (773-780).
d-fructose (10 mM) augments, in rat pancreatic islets, insulin release evoked by 10 mM d-glucose. Even in the absence of d-glucose, d-fructose (100 mM) displays a positive insulinotropic action. It was now examined whether the insulinotropic action of d-fructose could be attributed to an increase in the ATP content of islet cells. After 30–60 min incubation in the presence of d-glucose and/or d-fructose, the ATP and ADP content was measured by bioluminescence in either rat isolated pancreatic islets (total ATP and ADP) or the supernatant of dispersed rat pancreatic islet cells exposed for 30 s to digitonine (cytosolic ATP and ADP). d-fructose (10 and 100 mM) was found to cause a concentration-related decrease in the total ATP and ADP content and ATP/ADP ratio below the basal values found in islets deprived of exogenous nutrient. Moreover, in the presence of 10 mM d-glucose, which augmented both the total ATP content and ATP/ADP ratio above basal value, d-fructose (10 mM) also lowered these two parameters. The cytosolic ATP/ADP ratio, however, was increased in the presence of d-glucose and/or d-fructose. Under the present experimental conditions, a sigmoidal relationship was found between such a cytosolic ATP/ADP ratio and either 86Rb net uptake by dispersed islet cells or insulin release from isolated islets. These data provide, to our knowledge, the first example of a dramatic dissociation between changes in total ATP content or ATP/ADP ratio and insulin release in pancreatic islets exposed to a nutrient secretagogue. Nevertheless, the cationic and insulinotropic actions of d-glucose and/or d-fructose were tightly related to the cytosolic ATP/ADP ratio.
Keywords: Pancreatic islet; Dispersed islet cell; ATP and ADP; d-glucose and d-fructose;

In the field of photosynthetic water oxidation it has been under debate whether TyrosineZ (TyrZ) acts as a hydrogen or an electron acceptor from water. In the former concept, direct contact of TyrZ with substrate water has been assumed. However, there is no direct evidence for the interaction between TyrZ and substrate water in active Photosystem II (PSII), instead most experiments have been performed on inhibited PSII. Here, this problem is tackled in active PSII by combining low temperature EPR measurements and quantum chemistry calculations. EPR measurements observed that the maximum yield of TyrZ oxidation at cryogenic temperature in the S0 and S1 states was around neutral pH and was essentially pH-independent. The yield of TyrZ oxidation decreased at acidic and alkaline pH, with pKs at 4.7–4.9 and 7.7, respectively. The observed pH-dependent parts at low and high values of pH can be explained as due to sample inactivation, rather than active PSII. The reduction kinetics of TyrZ · in the S0 and S1 states were pH independent at pH range from 4.5 to 8. Therefore, the change of the pH in bulk solution probably has no effect on the TyrZ oxidation and TyrZ · reduction at cryogenic temperature in the S0 and S1 states of the active PSII. Theoretical calculations indicate that TyrZ becomes more difficult to oxidize when a H2O molecule interacts directly with it. It is suggested that TyrZ is probably located in a hydrophobic environment with no direct interaction with the substrate H2O in active PSII. These results provide new insights on the function and mechanism of water oxidation in PSII.
Keywords: Photosystem II; Tyrosine Z; EPR; Substrate water; pH dependence; DFT calculation;

The dark recovery kinetics of the Chl a fluorescence transient (OJIP) after 15 min light adaptation were studied and interpreted with the help of simultaneously measured 820 nm transmission. The kinetics of the changes in the shape of the OJIP transient were related to the kinetics of the qE and qT components of non-photochemical quenching. The dark-relaxation of the qE coincided with a general increase of the fluorescence yield. Light adaptation caused the disappearance of the IP-phase (20–200 ms) of the OJIP-transient. The qT correlated with the recovery of the IP-phase and with a recovery of the re-reduction of P700+ and oxidized plastocyanin in the 20–200 ms time-range as derived from 820 nm transmission measurements. On the basis of these observations, the qT is interpreted to represent the inactivation kinetics of ferredoxin-NADP+-reductase (FNR). The activation state of FNR affects the fluorescence yield via its effect on the electron flow. The qT therefore represents a form of photochemical quenching. Increasing the light intensity of the probe pulse from 1800 to 15000 μmol photons m−2 s−1 did not qualitatively change the results. The presented observations imply that in light-adapted leaves, it is not possible to ‘close’ all reaction centers with a strong light pulse. This supports the hypothesis that in addition to Q A a second modulator of the fluorescence yield located on the acceptor side of photosystem II (e.g., the occupancy of the Q B-site) is needed to explain these results. Besides, some of our results indicate that in pea leaves state 2 to 1 transitions may contribute to the qI-phase.
Keywords: Chl a fluorescence; qT; qN sv; OJIP-transient; 820 nm transmission; Pisum sativum;

To get an insight to the mechanism of the zeaxanthin-dependent non-photochemical quenching in photosystem II of photosynthesis, we probed the interaction of some xanthophylls with excited chlorophyll-a by trapping both pigments in micelles of triton X-100. Optimal distribution of pigments among micelles was obtained by proper control of the micelle concentration, using formamide in the reaction mixture, which varies the micellar aggregation number over three orders of magnitude. The optimal reaction mixture was obtained around 40% (v/v) formamide in 0.2–0.4% (v/v) triton X-100 in water. Zeaxanthin in the micellar solution exhibited initially absorption and circular dichroism spectral features corresponding to a J-type aggregate. The spectrum was transformed over time (half-time values vary—an average characteristic figure is roughly 20 min) to give features representing an H-type aggregate. The isosbestic point in the series of spectral curves favors the supposition of a rather simple reaction between two pure J and H-types dimeric species. Violaxanthin exhibited immediately stable spectral features corresponding to a mixture of J-type and more predominately H-type dimers. Lutein, neoxanthin and β-carotene did not show any aggregated spectral forms in micelles. The spectral features in micelles were compared to spectra in aqueous acetone, where the assignment to various aggregated types was established previously. The specific tendency of zeaxanthin to form the J-type dimer (or aggregate) could be important for its function in photosynthesis. The abilities of five carotenoids (zeaxanthin, violaxanthin, lutein, neoxanthin and β-carotene) to quench chlorophyll-a fluorescence were compared. Zeaxanthin, in its two micellar dimeric forms, and β-carotene were comparable good quenchers of chlorophyll-a fluorescence. Violaxanthin was a much weaker quencher, if at all. Lutein and neoxanthin rather enhanced the fluorescence. The implications to non-photochemical quenching process in photosynthesis are discussed.
Keywords: Triton X-100; Micelle; Micellar aggregation number; Formamide; β-carotene; Violaxanthin; Zeaxanthin; Lutein; Neoxanthin; Aggregate; Dimer; Circular dichroism; Concentration self-quenching;

Aggregation of the 636 nm emitting monomeric protochlorophyllide form into flash-photoactive, oligomeric 644 and 655 nm emitting forms in vitro by Annamária Kósa; Zsuzsanna Márton; Katalin Solymosi; Károly Bóka; Béla Böddi (811-820).
Artificial formation of flash-photoactive oligomeric protochlorophyllide complexes was found in etiolated pea (Pisum sativum L. cv. Zsuzsi) epicotyl homogenates containing glycerol (40% v/v) and sucrose (40% m/v). The 77 K fluorescence emission spectra indicated that the ratio of the 644 and 655 nm emitting forms to the 636 nm form increased during 3 to 5-day incubation in the dark at −14 °C. Electron micrographs showed the presence of well-organized prolamellar bodies in the homogenates. The same phenomena were found when the homogenates were frozen into liquid nitrogen and thawed to room temperature in several cycles. Similar treatments of intact epicotyl pieces caused significant membrane destructions. In homogenates, the in vitro produced 644 and 655 nm emitting protochlorophyllide forms were flash-photoactive; the extent of phototransformation increased compared to that in native epicotyls. The newly appeared 692 nm chlorophyllide band showed a blue shift (similar to the Shibata shift in leaves), however this process took place only partially due to the effect of the isolation medium.These results prove that the in vitro accumulated 644 and 655 nm protochlorophyllide forms were produced from the flash-photoactive 636 nm emitting monomeric NADPH:protochlorophyllide oxidoreductase units via aggregation, in connection with structure stabilization properties of glycerol and sucrose.
Keywords: Aggregation; Glycerol; NADPH:protochlorophyllide oxidoreductase (POR); Protochlorophyllide; Sucrose;

Putrescine stimulates chemiosmotic ATP synthesis by Nikolaos E. Ioannidis; Liliana Sfichi; Kiriakos Kotzabasis (821-828).
Putrescine is a main polyamine found in animals, plants and microbes, but the molecular mechanism underlying its mode of action is still obscure. In vivo chlorophyll a fluorescence in tobacco leaf discs indicated that putrescine treatment affects the energization of the thylakoid membrane. Molecular dissection of the electron transport chain by biophysical and biochemical means provided new evidence that putrescine can play an important bioenergetic role acting as a cation and as a permeant natural buffer. We demonstrate that putrescine increases chemiosmotic ATP synthesis more than 70%. Also a regulation of the energy outcome by small changes in putrescine pool under the same photonic environment (i.e., photosynthetically active radiation) is shown. The proposed molecular mechanism has at least four conserved features: (i) presence of a membrane barrier, (ii) a proton-driven ATPase, (iii) a ΔpH and (iv) a pool of putrescine.
Keywords: ATP; Chloroplast; Proton motive force; Putrescine; Internal buffering capacity;

Cyclic electron transfer in photosystem II in the marine diatom Phaeodactylum tricornutum by W. Onno Feikema; Marcell A. Marosvölgyi; Johann Lavaud; Hans J. van Gorkom (829-834).
In Phaeodactylum tricornutum Photosystem II is unusually resistant to damage by exposure to high light intensities. Not only is the capacity to dissipate excess excitations in the antenna much larger and induced more rapidly than in other organisms, but in addition an electron transfer cycle in the reaction center appears to prevent oxidative damage when secondary electron transport cannot keep up with the rate of charge separations. Such cyclic electron transfer had been inferred from oxygen measurements suggesting that some of its intermediates can be reduced in the dark and can subsequently compete with water as an electron donor to Photosystem II upon illumination. Here, the proposed activation of cyclic electron transfer by illumination is confirmed and shown to require only a second. On the other hand the dark reduction of its intermediates, specifically of tyrosine Y D, the only Photosystem II component known to compete with water oxidation, is ruled out. It appears that the cyclic electron transfer pathway can be fully opened by reduction of the plastoquinone pool in the dark. Oxygen evolution reappears after partial oxidation of the pool by Photosystem I, but the pool itself is not involved in cyclic electron transfer.
Keywords: Cyclic electron transfer; Photosystem II; Diatoms; Tyrosine D; Photo-inhibition;

The availability of the three dimensional structure of mitochondrial enzyme, obtained by X-ray crystallography, allowed a significant progress in the understanding of the structure–function relation of the cytochrome bc 1 complex. Most of the structural information obtained has been confirmed by molecular genetic studies of the bacterial complex. Despite its small size and simple subunit composition, high quality crystals of the bacterial complex have been difficult to obtain and so far, only low resolution structural data has been reported. The low quality crystal observed is likely associated in part with the low activity and stability of the purified complex. To mitigate this problem, we recently engineered a mutant [S287R(cytb)/V135S(ISP)] from Rhodobacter sphaeroides to produce a highly active and more stable cytochrome bc 1 complex. The purified mutant complex shows a 40% increase in electron transfer activity as compared to that of the wild type enzyme. Differential scanning calorimetric study shows that the mutant is more stable than the wild type complex as indicated by a 4.3 °C increase in the thermo-denaturation temperature. Crystals formed from this mutant complex, in the presence of stigmatellin, diffract X-rays up to 2.9 Å resolution.
Keywords: Cytochrome bc 1 complex;

Charge separation in photosystem II core complexes induced by 690–730 nm excitation at 1.7 K by Joseph L. Hughes; Paul Smith; Ron Pace; Elmars Krausz (841-851).
The illumination of oxygen-evolving PSII core complexes at very low temperatures in spectral regions not expected to excite P680 leads to charge separation in a majority of centers. The fraction of centers photoconverted as a function of the number of absorbed photons per PSII core is determined by quantification of electrochromic shifts on PheoD1. These shifts arise from the formation of metastable plastoquinone anion (QA ) configurations. Spectra of concentrated samples identify absorption in the 700–730 nm range. This is well beyond absorption attributable to CP47. Spectra in the 690–730 nm region can be described by the ‘trap’ CP47 absorption at 689 nm, with dipole strength of ∼1 chlorophyll a (chl a), partially overlapping a broader feature near 705 nm with a dipole strength of ∼0.15 chl a. This absorption strength in the 700–730 nm region falls by 40% in the photoconverted configuration. Quantum efficiencies of photoconversion following illumination in the 690–700 nm region are similar to those obtained with green illumination but fall significantly in the 700–730 nm range. Two possible assignments of the long-wavelength absorption are considered. Firstly, as a low intensity component of strongly exciton-coupled reaction center chlorin excitations and secondly as a nominally ‘dark’ charge-transfer excitation of the ‘special pair’ PD1–PD2. The opportunities offered by these observations towards the understanding of the nature of P680 and PSII fluorescence are discussed.
Keywords: Charge transfer; Reaction center; Exciton coupling; Photoconversion; Quantum efficiency;

The molecular mechanism for proton conduction in the D-pathway of Cytochrome c Oxidase (CcO) is investigated through the free energy profile, i.e., potential of mean force (PMF) calculations of both the native enzyme and the N98D mutant. The multistate empirical valence bond (MS-EVB) model was applied to simulate the interaction of an excess proton with the channel environment. In the study of the wild type enzyme, the PMF reveals the previously proposed proton trap inside the channel; it also shows a high free energy barrier against the passage of proton at the entry of the channel, where two conserved asparagines (ASN80/98) may be essential for the gating of proton uptake. We also present data from an investigation of the N98D mutant, which has been previously shown to completely eliminate proton pumping but significantly enhance the oxidase activity in Rhodobacter sphaeroides. These results suggest that mutating Asn98 to negatively charged aspartate will create an unfavorable energy barrier sufficiently high to prevent the overall proton uptake through the D-pathway, whereas with a protonated aspartic acid the proton conduction was found to be accelerated. Plausible explanations for the origin of the uncoupling of proton pumping from the oxidase activity will be discussed.
Keywords: Cytochrome c Oxidase; Multistate empirical valence bond (MS-EVB); Free energy calculation; Proton pumping;

Role of a putative third subunit YhcB on the assembly and function of cytochrome bd-type ubiquinol oxidase from Escherichia coli by Tatsushi Mogi; Eri Mizuochi-Asai; Sachiko Endou; Satoru Akimoto; Hiro Nakamura (860-864).
Recent proteome studies on the Escherichia coli membrane proteins suggested that YhcB is a putative third subunit of cytochrome bd-type ubiquinol oxidase (CydAB) (F. Stenberg, P. Chovanec, S.L. Maslen, C.V. Robinson, L.L. Ilag, G. von Heijne, D.O. Daley, Protein complexes of the Escherichia coli cell envelope. J. Biol. Chem. 280 (2005) 34409–34419). We isolated and characterized cytochrome bd from the ΔyhcB strain, and found that the formation of the CydAB heterodimer, the spectroscopic properties of bound hemes, and kinetic parameters for the ubiquinol-1 oxidation were identical to those of cytochrome bd from the wild-type strain. Anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis showed that YhcB was not associated with the cytochrome bd complex. We concluded that YhcB is dispensable for the assembly and function of cytochrome bd. YhcB, which is distributed only in γ-proteobacteria, may be a part of another membrane protein complex or may form a homo multimeric complex.
Keywords: Cytochrome bd; Membrane protein complex; Transposon mutant; Proteome; Ubiquinol oxidase; YhcB;