BBA - Bioenergetics (v.1460, #2-3)

New insight into the structure and function of the alternative oxidase by Deborah A Berthold; Martin E Andersson; Pär Nordlund (241-254).
The alternative oxidase is a ubiquinol oxidase found in plant mitochondria, as well as in the mitochondria of some fungi and protists. It catalyzes a cyanide-resistant reduction of oxygen to water without translocation of protons across the inner mitochondrial membrane, and thus functions as a non-energy-conserving member of the respiratory electron transfer chain. The active site of the alternative oxidase has been modelled as a diiron center within a four-helix bundle by Siedow et al. (FEBS Lett. 362 (1995) 10–14) and more recently by Andersson and Nordlund (FEBS Lett. 449 (1999) 17–22). The cloning of the Arabidopsis thaliana IMMUTANS (Im) gene, which encodes a plastid enzyme distantly related to the mitochondrial alternative oxidases (Wu et al. Plant Cell 11 (1999) 43–55; Carol et al. Plant Cell 11 (1999) 57–68), has now narrowed the range of possible ligands to the diiron center of the alternative oxidase. The Im protein sequence suggests a minor modification to the recent model of the active site of the alternative oxidase. This change moves an invariant tyrosine into a conserved hydrophobic pocket in the vicinity of the active site, in a position analogous to the long-lived tyrosine radical at the diiron center of ribonucleotide reductase, and similar to the tyrosines near the diiron center of bacterioferritin and rubrerythrin. The Im sequence and modified structural model yield a compelling picture of the alternative oxidase as a diiron carboxylate protein. The current status of the relationship of structure to function in the alternative oxidase is reviewed.
Keywords: Alternative oxidase; Ubiquinol oxidase; Plastoquinol oxidase; IMMUTANS; Carotenoid biosynthesis; Diiron protein;

Light response curves of photosystem (PS) II electron transport from oxygen evolving complex to plastoquinone (PQ) were measured in sunflower (Helianthus annuus L.), cotton (Gossypium hirsutum L.) and tobacco (Nicotiana tabacum L.) leaves by recording O2 evolution and fluorescence in 5–200 ms light pulses of 500–13 500 μmol absorbed quanta m−2 s−1. The leaves were pre-adapted at 60–2000 μmol quanta m−2 s−1 for 60 min to obtain different nonphotochemical excitation quenching, which was predominantly of reversible q I type (relaxation time 30 min). PQ was completely oxidized by turning the actinic light off and illuminating with far-red light for 2 s before the pulse was applied in the dark, 4 s after the actinic light was turned off. Electron transport rate calculated from fluorescence transients considering PS II donor side resistance (V. Oja, A. Laisk, submitted) was maximal at the beginning of pulses (J Fi) and decreased immediately. The dependences of J Fi on pulse absorbed flux density were rectangular hyperbolas with K m about 7500 μmol m−2 s−1. Both the extrapolated plateau J Fm and initial slope (intrinsic quantum yield of PS II, Y m) decreased proportionally when q I increased from minimum to maximum (J Fm from 2860 to 1450 μmol e m−2 s−1 and Y m from 0.41 to 0.23). The time constant for electron transfer away from the PS II acceptor side, calculated from a model of PS II electron transport for 2 μmol PS II m−2, increased from 607 to 1315 μs with the activation of q I while the donor side time constant changed from 289 to 329 μs. These results show that changes in the electron transfer processes on the acceptor side of PS II occur in parallel with nonphotochemical (predominantly reversible q I type) excitation quenching.
Keywords: Photosynthesis; Photosystem II; Nonphotochemical quenching;

Are mitochondria a permanent source of reactive oxygen species? by Katrin Staniek; Hans Nohl (268-275).
The observation that in isolated mitochondria electrons may leak out of the respiratory chain to form superoxide radicals (O2 •−) has prompted the assumption that O2 •− formation is a compulsory by-product of respiration. Since mitochondrial O2 •− formation under homeostatic conditions could not be demonstrated in situ so far, conclusions drawn from isolated mitochondria must be considered with precaution. The present study reveals a link between electron deviation from the respiratory chain to oxygen and the coupling state in the presence of antimycin A. Another important factor is the analytical system applied for the detection of activated oxygen species. Due to the presence of superoxide dismutase in mitochondria, O2 •− release cannot be realistically determined in intact mitochondria. We therefore followed the release of the stable dismutation product H2O2 by comparing most frequently used H2O2 detection methods. The possible interaction of the detection systems with the respiratory chain was avoided by a recently developed method, which was compared with conventional methods. Irrespective of the methods applied, the substrates used for respiration and the state of respiration established, intact mitochondria could not be made to release H2O2 from dismutating O2 •−. Although regular mitochondrial respiration is unlikely to supply single electrons for O2 •− formation our study does not exclude the possibility of the respiratory chain becoming a radical source under certain conditions.
Keywords: Mitochondrion; Heart; Superoxide radical; Hydrogen peroxide; Fluorescence; Scopoletin; Homovanillic acid;

An elementary kinetic model of energy coupling in biological membranes by Ernesto Cristina; Julio A Hernández (276-290).
The purpose of this work is to contribute to the understanding of the fundamental kinetic properties of the processes of energy coupling in biological membranes. For this, we consider a model of a microorganism that, in its plasma membrane, expresses two electrogenic enzymes (E1 and E2) transporting the same monovalent cation C and electrodiffusive paths for C and for a monovalent anion A. E1 (E2) couples transport C to the reaction S1↔P1 (S2↔P2). We developed a mathematical model that describes the rate of change of the electrical potential difference across the membrane, of the internal concentrations of C and A, and of the concentrations of S2 and P2. The enzymes are incorporated via two-state kinetic models; the passive ionic fluxes are represented by classical formulations of electrodiffusion. The microorganism volume is maintained constant by accessory regulatory devices. The model is utilized for stationary and dynamic studies for the case of bacteria employing the electrochemical gradient of Na+ as energetic intermediate. Among other conclusions, the results show that the membrane potential represents the relevant kinetic intermediate for the overall coupling between the energy donor reaction S1↔P1 and the synthesis of S2.
Keywords: Membrane; Energy coupling; Ion pump; Mathematical model;

O2 evolution from single turnover flashes of up to 96 μmol absorbed quanta m−2 and from multiple turnover pulses of 8.6 and 38.6 ms duration and 12 800 and 850 μmol absorbed quanta m−2 s−1 intensity, respectively, was measured in sunflower leaves with the help of zirconium O2 analyser. O2 evolution from one flash could be measured with 1% accuracy on the background of 10–50 μmol O2 mol−1. Before the measurements leaves were pre-adapted either at 30–60 or 1700 μmol quanta m−2 s−1 to induce different non-photochemical excitation quenching (q N). Short (1 min) exposures at the high light that created only energy-dependent, q E type quenching, caused no changes in the O2 yield from saturating flashes or pulses that could be related to the q E quenching, but the yield from low intensity flashes and pulses decreased considerably. Long 30–60-min exposures at the high light induced a reversible inhibitory, q I type quenching that decreased the O2 yield from both, saturating and limiting flashes and pulses (but more from the limiting ones), which reversed within 15 min under the low light. The results are in agreement with the notion that q E is caused by a quenching process in the PSII antenna and no changes occur in the PSII centres, but the reversible (15–30 min) q I quenching is accompanied by inactivation of a part of PSII centres.
Keywords: Leaf; Photosynthesis; Photosystem II; Excitation quenching;

Definition of crucial structural factors of acetogenins, potent inhibitors of mitochondrial complex I by Motoyuki Takada; Kaoru Kuwabara; Hironori Nakato; Akira Tanaka; Hajime Iwamura; Hideto Miyoshi (302-310).
Some natural acetogenins are the most potent inhibitors of bovine heart mitochondrial complex I. These compounds are characterized by two functional units (i.e. hydroxylated tetrahydrofuran (THF) and α,β-unsaturated γ-lactone ring moieties) separated by a long alkyl spacer. To elucidate which structural factors of acetogenins including their active conformation are crucial for the potent inhibitory effect, we synthesized a series of novel acetogenin analogues possessing bis-THF rings. The present study clearly demonstrated that the natural γ-lactone ring is not crucial for the potent inhibition, although this moiety is the most common structural unit among a large number of natural acetogenins and has been suggested to be the only reactive species that directly interacts with the enzyme (Shimada et al., Biochemistry 37 (1998) 854–866). The presence of free hydroxy group(s) in the adjacent bis-THF rings was favorable, but not essential, for the potent activity. This was probably because high polarity (or hydrophilicity), rather than hydrogen bond-donating ability, around the bis-THF rings is required to retain the inhibitor in the active conformation. Interestingly, length of the alkyl spacer proved to be a very important structural factor for the potent activity, the optimal length being approximately 13 carbon atoms. The present study provided further strong evidence for the previous proposal (Kuwabara et al., Eur. J. Biochem. 267 (2000) 2538–2546) that the γ-lactone and THF ring moieties act in a cooperative manner on complex I with the support of some specific conformation of the spacer.
Keywords: Respiratory enzyme; Mitochondrial complex I; Acetogenin; Structure–activity relationship;

Isolated chloroplasts show a light-induced reversible increase in blue-green fluorescence (BGF), which is only dependent on NADPH changes. In the present communication, we report a time-resolved and spectral analysis of this BGF in reconstituted chloroplasts and intact isolated chloroplasts, in the dark and under actinic illumination. From these measurements we deduced the contribution of the different forms of NADPH (free and bound to proteins) to the light-induced variation of BGF and conclude that this variation is due only to the redox change of the NADP pool. A simple model estimating the distribution of NADPH between the free and bound form was designed, that explains the differences measured for the BGF of reconstituted chloroplasts and intact chloroplasts. From the decay-associated spectra of the chloroplast BGF, we also deduced the participation of flavins to the green peak of chloroplast fluorescence emission spectrum, and the existence of excitation energy transfer from proteins to bound NADPH in chloroplasts. In addition, we re-examined the use of chloroplast BGF as a quantitative measure of NADPH concentration, and confirmed that chloroplast BGF can be used for non-destructive, continuous and probably quantitative monitoring of light-induced changes in NADP redox state.
Keywords: Blue-green fluorescence; Decay-associated spectra; Excitation energy transfer; Flavin; Intact chloroplast; Reconstituted chloroplast; Pyridine nucleotide;

The electron–electron double resonance (ELDOR) method was applied to measure the dipole interaction between cytochrome (Cyt) b + 559 and the primary acceptor quinone (Q A), observed at g=2.0045 with the peak to peak width of about 9 G, in Photosystem II (PS II) in which the non-heme Fe2+ was substituted by Zn2+. The paramagnetic centers of Cyt b + 559Y DQ A were trapped by illumination at 273 K for 8 min, followed by dark adaptation for 3 min and freezing into 77 K. The distance between the pair Cyt b + 559-Q A was estimated from the dipole interaction constant fitted to the observed ELDOR time profile to be 40±1 Å. In the membrane oriented PS II particles the angle between the vector from QA to Cyt b 559 and the membrane normal was determined to be 80±5°. The position of Cyt b 559 relative to QA suggests that the heme plane is located on the stromal side of the thylakoid membrane. ELDOR was not observed for Cyt b + 559 Y D spin pair, suggesting the distance between them is more than 50 Å.
Keywords: Photosystem II; Oriented membrane; Cytochrome b 559; QA; Electron spin echo;

Energy transfer and charge separation in the purple non-sulfur bacterium Roseospirillum parvum by Hjalmar P. Permentier; Sieglinde Neerken; Kristiane A. Schmidt; Jörg Overmann; Jan Amesz (338-345).
The antenna reaction centre system of the recently described purple non-sulfur bacterium Roseospirillum parvum strain 930I was studied with various spectroscopic techniques. The bacterium contains bacteriochlorophyll (BChl) a, 20% of which was esterified with tetrahydrogeranylgeraniol. In the near-infrared, the antenna showed absorption bands at 805 and 909 nm (929 nm at 6 K). Fluorescence bands were located at 925 and 954 nm, at 300 and 6 K, respectively. Fluorescence excitation spectra and time resolved picosecond absorbance difference spectroscopy showed a nearly 100% efficient energy transfer from BChl 805 to BChl 909, with a time constant of only 2.6 ps. This and other evidence indicate that both types of BChl belong to a single LH1 complex. Flash induced difference spectra show that the primary electron donor absorbs at 886 nm, i.e. at 285 cm−1 higher energy than the long wavelength antenna band. Nevertheless, the time constant for trapping in the reaction centre was the same as for almost all other purple bacteria: 55±5 ps. The shape as well as the amplitude of the absorbance difference spectrum of the excited antenna indicated exciton interaction and delocalisation of the excited state over the BChl 909 ring, whereas BChl 805 appeared to have a monomeric nature.
Keywords: Antenna complex; Charge separation; Energy transfer; Light harvesting complex 1; Purple bacterium;

Resistance of isolated pulmonary mitochondria during in vitro anoxia/reoxygenation by Katty Willet; Olivier Detry; Francis E. Sluse (346-352).
The aim of the study was to investigate the effect of in vitro anoxia/reoxygenation on the oxidative phosphorylation of isolated lung mitochondria. Mitochondria were isolated after harvesting from fresh pig lungs flushed with Euro-Collins solution. Mitochondrial respiratory parameters were determined in isolated mitochondria before anoxia (control), after 5–45 min anoxia followed by 5 min reoxygenation, and after 25 or 40 min of in vitro incubation in order to follow the in vitro aging of mitochondria during respiratory assays. Respiratory parameters measured after anoxia/reoxygenation did not show any oxidative phosphorylation dysfunction, indicating a high resistance of pulmonary mitochondria to in vitro anoxia/reoxygenation (up to 45 min anoxia). These results indicate that mitochondria are not directly responsible of their oxidative phosphorylation damage observed after in vivo ischemia (K. Willet et al., Transplantation 69 (2000) 582) but are a target of others cellular injuries leading to mitochondrial dysfunction in vivo.
Keywords: Mitochondria; Anoxia; Reoxygenation; Oxidative phosphorylation; Transplantation; (Lung);

The various types of redox partner interactions employed in cytochrome P450 systems are described. The similarities and differences between the redox components in the major categories of P450 systems present in bacteria, mitochondria and microsomes are discussed in the light of the accumulated evidence from X-ray crystallographic and NMR spectroscopic determinations. Molecular modeling of the interactions between the redox components in various P450 mono-oxygenase systems is proposed on the basis of structural and mutagenesis information, together with experimental findings based on chemical modification of key residues likely to be associated with complementary binding sites on certain typical P450 isoforms and their respective redox partners.
Keywords: Cytochrome P450; Redox partner interaction; Electron transfer rate;

A lysine residue involved in the inhibition of vacuolar H+-pyrophosphatase by fluorescein 5′-isothiocyanate by Su Jing Yang; Shih Sheng Jiang; Ru Chuan Van; Yi Yuong Hsiao; Rong-Long Pan (375-383).
Vacuolar proton pumping pyrophosphatase (H+-PPase; EC 3.6.1.1) plays a central role in the electrogenic translocation of protons from cytosol to the vacuole lumen at the expense of PPi hydrolysis. A fluorescent probe, fluorescein 5′-isothiocyanate (FITC), was used to modify a lysine residue of vacuolar H+-PPase. The enzymatic activity and its associated H+ translocation of vacuolar H+-PPase were markedly decreased by FITC in a concentration-dependent manner. The inhibition of enzymatic activity followed pseudo-first-order rate kinetics. A double-logarithmic plot of the apparent reaction rate constant against FITC concentration yielded a straight line with a slope of 0.89, suggesting that the alteration of a single lysine residue on the enzyme is sufficient to inhibit vacuolar H+-PPase. Changes in K m but not V max values of vacuolar H+-PPase as inhibited by FITC were obtained, indicating that the labeling caused a modification in affinity of the enzyme to its substrate. FITC inhibition of vacuolar H+-PPase could be protected by its physiological substrate, Mg2+-PPi. These results indicate that FITC might specifically compete with the substrate at the active site and the FITC-labeled lysine residue locates probably in or near the catalytic domain of the enzyme. The enhancement of fluorescence intensity and the blue shift of the emission maximum of FITC after modification of vacuolar H+-PPase suggest that the FITC-labeled lysine residue is located in a relatively hydrophobic region.
Keywords: Tonoplast; H+-pyrophosphatase; Fluorescein 5′-isothiocyanate;

Kinetic characterization of His-tagged CP47 Photosystem II in Synechocystis sp. PCC6803 by Zhao-Liang Li; Terry M. Bricker; Robert Burnap (384-389).
Recently, construction of strains of Synechocystis sp. PCC6803 having a His6 extension (His-tag) of the carboxyl terminus of the CP47 protein has been reported (T.M. Bricker et al, Biochim. Biophys. Acta 1409 (1998) 50; M.J. Reifler et al., in: Garab, Pusztai (Eds.) Proc. XIth International Congress on Photosynthesis, 1998). While these initial reports suggest a minimal impact of the His-tag upon Photosystem (PS) II function, a more thorough analysis of the kinetic properties of the modified complex is essential. This communication reports on a more detailed kinetic analysis to assess possible perturbations of PS II due to the genetic addition of the His-tag on the CP47 protein. It was found that: (1) Patterns of flash O2 yield exhibited normal period four oscillations and the associated fits of the Kok-Joliot S-state cycling parameters were virtually identical to the wild type; (2) O2 release kinetics during the S3–S0 transition were experimentally indistinguishable from the wild type; (3) S-state decay measurements indicate slightly faster decays of the S2 and S3 states compared to the wild type; (4) fluorescence measurements indicate that the kinetics of the forward reaction of electron transfer from QA to QB and back-reactions of QA with PS II electron donors are similar in the His-tag and wild-type strains. It is therefore concluded that the addition of the His-tag results in a minimal perturbation of PS II function.
Keywords: Photosynthesis; Oxygen evolution; Affinity purification; Histidine tagging;