BBA - Bioenergetics (v.1554, #1-2)

The photochemical activity of NADPH:protochlorophyllide oxidoreductase (POR) was studied in etiolated wheat (Triticum aestivum, L., cult. MV17) leaf homogenates. The kinetics of the transformation of protochlorophyllide into chlorophyllide was detected by fluorescence intensity changes at 690 nm (formation of chlorophyllide) and 655 nm (decay of protochlorophyllide) at 20 °C, excited at 440 nm while the pressure was varied between 0.1 and 400 MPa. Both kinetics could be fitted by two exponentials and the reaction rates were pressure-dependent. A model was suggested based on the comparison of the two kinetics. Reaction rates of the processes occurring during the prototransformation were determined in function of pressure. The evaluation yielded the activation volume as 1.7 ml mol−1, which corresponds with the formation of one H-bond/molecule.
Keywords: Protochlorophyllide; Chlorophyllide; Fluorescence; High pressure fluorescence spectroscopy; Activation volume;

Structure–function relationships in Anabaena ferredoxin/ferredoxin:NADP+ reductase electron transfer: insights from site-directed mutagenesis, transient absorption spectroscopy and X-ray crystallography by John K. Hurley; Renaud Morales; Marta Martı́nez-Júlvez; Tammy B. Brodie; Milagros Medina; Carlos Gómez-Moreno; Gordon Tollin (5-21).
The interaction between reduced Anabaena ferredoxin and oxidized ferredoxin:NADP+ reductase (FNR), which occurs during photosynthetic electron transfer (ET), has been investigated extensively in the authors' laboratories using transient and steady-state kinetic measurements and X-ray crystallography. The effect of a large number of site-specific mutations in both proteins has been assessed. Many of the mutations had little or no effect on ET kinetics. However, non-conservative mutations at three highly conserved surface sites in ferredoxin (F65, E94 and S47) caused ET rate constants to decrease by four orders of magnitude, and non-conservative mutations at three highly conserved surface sites in FNR (L76, K75 and E301) caused ET rate constants to decrease by factors of 25–150. These residues were deemed to be critical for ET. Similar mutations at several other conserved sites in the two proteins (D67 in Fd; E139, L78, K72, and R16 in FNR) caused smaller but still appreciable effects on ET rate constants. A strong correlation exists between these results and the X-ray crystal structure of an Anabaena ferredoxin/FNR complex. Thus, mutations at sites that are within the protein–protein interface or are directly involved in interprotein contacts generally show the largest kinetic effects. The implications of these results for the ET mechanism are discussed.
Keywords: Protein–protein interaction; Kinetic; Photosynthetic electron transfer; Transient complex; X-ray crystal structure;

Intramolecular proton transfer of heme-copper oxidases is performed via the K- and the transmembrane D-channels. A carboxyl group conserved in a subgroup of heme-copper oxidases, located within the D-channel close to the binuclear center (=glutamic acid-286 in cytochrome bo 3 from Escherichia coli) is essential for proton pumping. Upon electron transfer to the fully oxidized (FO) enzyme, this amino acid has been shown to undergo a cyanide-independent environmental change. The redox-induced environmental transition of glutamic acid-286 is preserved in the site-directed mutant Y288F, which has lost its CuB binding capacity. Furthermore, the mixed-valence (MV) redox state of cytochrome bo 3 (in which CuB and high-spin heme are reduced, whereas the low-spin heme stays oxidized) was prepared by anaerobic exposure of the protein to carbon monoxide. This complex was converted (i) to the FO state by reaction with the caged dioxygen donor (μ-peroxo) (μ-hydroxo) bis [bis (bipyridyl) cobalt (III)] and (ii) to the fully reduced (FR) state via caged electron donors; the environmental change of glutamic acid-286 could be observed only upon reduction. Taken together, these results from two different lines of evidence clearly show that the redox transition of the low-spin heme b center alone triggers the change in the chemical environment of this acidic side chain. It is suggested that glutamic acid-286 is a kinetic enhancer of proton translocation, which is energetically favoured in mesophilic oxidases.
Keywords: Heme-copper oxidase; Cytochrome bo 3; Mixed valence; Carbon monoxide; Caged compound; FT-IR spectroscopy;

Photosynthetic oxygen evolution is an extremely heat-sensitive process and incubation of spinach Photosystem II (PSII) membranes at 40 °C for only several minutes leads to its complete inactivation. Substitution experiments of the spinach 33-kDa manganese stabilizing protein by a homologue protein, isolated either from the thermophilic cyanobacterium Phormidium laminosum, or from Escherichia coli as a recombinant thermophilic cyanobacterial protein, showed a significant increase in tolerance to heat inactivation of the oxygen-evolving activity. The results allow us to suggest that thermal inactivation of oxygen evolution in higher plant PSII membranes is due to dissociation of the 33-kDa protein as a consequence of temperature-induced conformational changes, and stabilization can be provided by substitution by a thermostable homologue whose secondary structure and binding to PSII remain unaltered at moderately high temperatures.
Keywords: Photosystem II; Heat inactivation; 33-kDa protein; Manganese stabilizing protein; Oxygen evolution;

Selective perturbation of the second electron transfer step in mutant bacterial reaction centers by Selma Schenkl; Sebastian Spörlein; Frank Müh; Heike Witt; Wolfgang Lubitz; Wolfgang Zinth; Josef Wachtveitl (36-47).
In order to specifically perturb the primary electron acceptor BA — a monomeric bacteriochlorophyll (BChl) a — involved in bacterial photosynthetic charge separation (CS), the protein environment of BA in the reaction center (RC) of Rhodobacter sphaeroides was modified by site-directed mutagenesis. Isolated RCs were characterized by redox titrations, low temperature optical spectroscopy, ENDOR/TRIPLE resonance spectroscopy and femtosecond time-resolved spectroscopy. Two mutations were studied: In the GS(M203) mutant a serine is introduced near the ring E keto group of BA, while in FY(L146) a phenylalanine near the ring A acetyl group of BA is replaced by tyrosine. In all mutations the oxidation potential of the primary electron donor P as well as the electronic structure of both the P •+ radical cation and the radical anion of the secondary electron acceptor, HA •−, are not significantly altered compared to the wild type (WT), while changes of the optical absorption spectra at 77 K in the BChl QX and QY regions are observed. The GS(M203) mutation only leads to a minor retardation of the CS reactions at room temperature, whereas for FY(L146) significant deviations from the native electron transfer (ET) rates could be detected: In addition to a faster first (2.9 ps) and a slower second (1 ps) ET step, a new 8-ps time constant was found in the FY(L146) mutant, which can be ascribed to a fraction of RCs with slowed down secondary ET. The results allow us to address the functional role of the acetyl group of BA and question the role of the free energy changes as the main determining factor of ET rates in RCs. It is concluded that structural rearrangements alter the electronic coupling between the pigments and thereby influence the rate of fast CS.
Keywords: Bacterial photosynthesis; Femtosecond spectroscopy; Electron nuclear double resonance; Primary electron transfer; Site-directed mutagenesis; Bacteriochlorophyll;

The quantitation of ADP diffusion gradients across the outer membrane of heart mitochondria in the presence of macromolecules by Frank Norbert Gellerich; Fanny Dorine Laterveer; Stephan Zierz; Klaas Nicolay (48-56).
We have previously provided evidence that diffusion of metabolites across the porin pores of mitochondrial outer membrane is hindered. A functional consequence of this diffusion limitation is the dynamic compartmentation of ADP in the intermembrane space. These earlier studies were done on isolated mitochondria suspended in isotonic media without macromolecules, in which intermembrane space of mitochondria is enlarged. The present study was undertaken to assess the diffusion limitation of outer membrane in the presence of 10% (w/v) dextran M20, in order to mimick the action of cytosolic macromolecules on mitochondria. Under these conditions, mitochondria have a more native, condensed configuration.Flux-dependent concentration gradients of ADP were estimated by measuring the ADP diffusion fluxes across the porin pores of isolated rat heart mitochondria incubated together with pyruvate kinase (PK), both of which compete for ADP regenerated by mitochondrial creatine kinase (mtCK) within the intermembrane space or by yeast hexokinase (HK) extramitochondrially. From diffusion fluxes and bulk phase concentrations of ADP, its concentrations in the intermembrane space were calculated using Fick's law of diffusion. Flux-dependent gradients up to 23 μM ADP (for a diffusion rate of J Dif=1.9 μmol ADP/min/mg mitochondrial protein) were observed. These gradients are about twice those estimated in the absence of dextran and in the same order of magnitude as the cytosolic ADP concentration (30 μM), but they are negligibly low for cytosolic ATP (5 mM). Therefore, it is concluded that the dynamic ADP compartmentation is of biological importance for intact heart cells.If mtCK generates ADP within the intermembrane space, the local ADP concentration can be clearly higher than in the cytosol resulting in higher extramitochondrial phosphorylation potentials. In this way, mtCK contributes to ensure optimal kinetic conditions for ATP-splitting reactions in the extramitochondrial compartment.
Keywords: Macromolecule; Oxidative phosphorylation; Creatine kinase; Hexokinase; Compartmentation; Concentration gradient; Rat;

Bcl-2 protects against apoptosis induced by antimycin A and bongkrekic acid without restoring cellular ATP levels by Aniek O. de Graaf; Jules P.P. Meijerink; Lambert P. van den Heuvel; Ronney A. DeAbreu; Theo de Witte; Joop H. Jansen; Jan A.M. Smeitink (57-65).
Several studies indicate that mitochondrial ATP production as well as ADP/ATP exchange across mitochondrial membranes are impaired during apoptosis. We investigated whether Bcl-2 could protect against cell death under conditions in which ATP metabolism is inhibited. Inhibition of ATP production using antimycin A (AA) (complex III inhibition) combined with inhibition of ADP/ATP exchange by bongkrekic acid (BA) (adenine nucleotide translocator (ANT) inhibition) induced a sharp decrease in total cellular ATP in FL5.12 parental cells (to 35% of untreated controls after 24 h of incubation). Within 24 and 48 h, 38% and 75% of the cells had died, respectively. However, in stably transfected FL5.12 Bcl-2 subclones, no cell death occurred under these experimental conditions. Similar results were obtained with Jurkat and Bcl-2 overexpressing Jurkat cells. Total cellular ATP levels were equally affected in FL5.12 Bcl-2 overexpressing cells and FL5.12 parental cells. This indicates that Bcl-2 overexpressing cells are able to survive with very low cellular ATP content. Furthermore, Bcl-2 did not protect against cell death by restoring ATP levels. This suggests that, under these conditions, Bcl-2 acts by inhibiting the signalling cascade triggered by the inhibitors that would normally lead to apoptosis.
Keywords: Bcl-2; ATP; Mitochondrion; Apoptosis; Adenine nucleotide translocator; Voltage-dependent anion channel;

HoxE—a subunit specific for the pentameric bidirectional hydrogenase complex (HoxEFUYH) of cyanobacteria by Oliver Schmitz; Gudrun Boison; Heike Salzmann; Hermann Bothe; Kathrin Schütz; Shu-hua Wang; Thomas Happe (66-74).
NAD(P)+-reducing hydrogenases have been described to be composed of a diaphorase (HoxFU) and a hydrogenase (HoxYH) moiety. This study presents for the first time experimental evidence that in cyanobacteria, a fifth subunit, HoxE, is part of this bidirectional hydrogenase. HoxE exhibits sequence identities to NuoE of respiratory complex I of Escherichia coli. The subunit composition of the cyanobacterial bidirectional hydrogenase has been investigated. The oxygen labile enzyme complex was purified to close homogeneity under anaerobic conditions from Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 6301. The 647-fold and 1290-fold enriched purified enzyme has a specific activity of 46 μmol H2 evolved (min mg protein)−1 and 15 μmol H2 evolved (min mg protein)−1, respectively. H2-evolution of the purified enzyme of S. sp. PCC 6803 is highest at 60 °C and pH 6.3. Immunoblot experiments, using a polyclonal anti-HoxE antibody, demonstrate that HoxE co-purifies with the hydrogenase activity in S. sp. PCC 6301. SDS-PAGE gels of the purified enzymes revealed six proteins, which were partially sequenced and identified, besides one nonhydrogenase component, as HoxF, HoxU, HoxY, HoxH and, remarkably, HoxE. The molecular weight of the native protein (375 kDa) indicates a dimeric assembly of the enzyme complex, Hox(EFUYH)2.
Keywords: Cyanobacteria; Bidirectional hydrogenase; Hydrogen metabolism; HoxE; NADH dehydrogenase;

Tuning of the optical and electrochemical properties of the primary donor bacteriochlorophylls in the reaction centre from Rhodobacter sphaeroides: spectroscopy and structure by Diane Spiedel; Aleksander W. Roszak; Kimberley McKendrick; Katherine E. McAuley; Paul K. Fyfe; Eliane Nabedryk; Jacques Breton; Bruno Robert; Richard J. Cogdell; Neil W. Isaacs; Michael R. Jones (75-93).
A series of mutations have been introduced at residue 168 of the L-subunit of the reaction centre from Rhodobacter sphaeroides. In the wild-type reaction centre, residue His L168 donates a strong hydrogen bond to the acetyl carbonyl group of one of the pair of bacteriochlorophylls (BChl) that constitutes the primary donor of electrons. Mutation of His L168 to Phe or Leu causes a large decrease in the mid-point redox potential of the primary electron donor, consistent with removal of this strong hydrogen bond. Mutations to Lys, Asp and Arg cause smaller decreases in redox potential, indicative of the presence of weak hydrogen bond and/or an electrostatic effect of the polar residue. A spectroscopic analysis of the mutant complexes suggests that replacement of the wild-type His residue causes a decrease in the strength of the coupling between the two primary donor bacteriochlorophylls. The X-ray crystal structure of the mutant in which His L168 has been replaced by Phe (HL168F) was determined to a resolution of 2.5 Å, and the structural model of the HL168F mutant was compared with that of the wild-type complex. The mutation causes a shift in the position of the primary donor bacteriochlorophyll that is adjacent to residue L168, and also affects the conformation of the acetyl carbonyl group of this bacteriochlorophyll. This conformational change constitutes an approximately 27° through-plane rotation, rather than the large into-plane rotation that has been widely discussed in the context of the HL168F mutation. The possible structural basis of the altered spectroscopic properties of the HL168F mutant reaction centre is discussed, as is the relevance of the X-ray crystal structure of the HL168F mutant to the possible structures of the remaining mutant complexes.
Keywords: Bacteriophyll; Rhodobacter sphaeroides; Spectroscopy;

Upon sudden exposure of plants to an actinic light of saturating intensity, the yield of chlorophyll fluorescence increases typically by 200–400% of the initial O-level. At least three distinct phases of these O–J–I–P transients can be resolved: O–J (0.05–5 ms), J–I (5–50 ms), and I–P (50–1000 ms). In thylakoid membranes, the J–I increase accounts for ∼30% of the total fluorescence increase; in Photosystem II membranes, the J–I phase is always lacking. In the presence of the ionophore valinomycin, which is known to inhibit specifically the formation of membrane voltages, the magnitude of the J–I phase is clearly diminished; in the presence of valinomycin supplemented by potassium, the J–I phase is fully suppressed. We conclude that the light-driven formation of the thylakoid-membrane voltage results in an increase of the chlorophyll excited-state lifetime, a phenomenon explainable by the electric-field-induced shift of the free-energy level of the primary radical pair [Dau and Sauer, Biochim. Biophys. Acta 1102 (1992) 91]. The assignment of the J–I increase in the fluorescence yield enhances the potential of using O–J–I–P fluorescence transients for investigations on photosynthesis in intact organisms. A putative role of thylakoid voltages in protection of PSII against photoinhibitory damage is discussed.
Keywords: Electric field effect; Fluorescence induction; Photosynthesis; Photosystem II; Variable fluorescence;

Eukaryotic cytochrome oxidases are composed of up to 13 subunits, of which three, subunits 1, 2 and 3, are mitochondrially encoded. In this study, yeast mutants were used to investigate the role of subunits 1 and 3 domains on the enzyme assembly. Mutation S203L in subunit 3 which abolished the respiratory growth, decreased cytochrome oxidase content, as measured by optical spectroscopy and immunodetection. Secondary mutations in subunits 1 and 3 restored (partly) the enzyme level. Two reversions reintroduced residues with a hydroxyl group at the primary mutation site (S203T) or in a subunit 3 transmembrane helix close to subunit 1 (G104S). These residues may be involved in hydrogen bonding which strengthen subunits 1–3 interaction. Two other reversions (A224V and Q137K) are located in P-side loops in subunit 1, which may be involved in the enzyme assembly. A mutation in residue A224 has been reported in a family presenting with encephalomyopathy. Surprisingly, the introduction of the ‘human’ mutation A224S and of a more drastic change A224F had no effect on the yeast enzyme. This might be explained by differences in local folding in the two enzymes.
Keywords: Cytochrome oxidase; Yeast; Assembly;

Conformational rearrangements in light-harvesting complex II accompanying light-induced chlorophyll a fluorescence quenching by Wojtek Grudziński; Zbigniew Krupa; Maciej Garstka; Waldemar Maksymiec; Trevor E. Swartz; Wiesław I. Gruszecki (108-117).
Light-induced chlorophyll a (Chl a) fluorescence quenching was studied in light-harvesting complex of photosystem II (LHCII). Fluorescence intensity decreased by ca. 20% in the course of 20 min illumination (412 nm, 36 μmol m−2 s−1) and was totally reversible within 30 min dark adaptation. The pronounced quenching was observed only in LHCII in an aggregated form and exclusively in the presence of molecular oxygen. Structural rearrangement of LHCII correlated to the quenching was monitored by measuring changes in UV–Visible light absorption spectra, and by measuring Fourier-transform infrared spectroscopy (FTIR) in the Amide I region of the protein (1600–1700 cm−1). The light-induced structural rearrangement of LHCII was interpreted as a partial disaggregation of the complex based on the decrease in the light scattering signal and the characteristic features observed in the FTIR spectra: the relative increase in the intensity of the band at 1653 cm−1, corresponding to a protein in the α-helical structure at the expense of the band centered at 1621 cm−1, characteristic of aggregated forms. The fact that the light-driven isomerization of the all-trans violaxanthin to the 13-cis form was not observed under the non-oxygenic conditions coincided with the lack of large-scale conformational reorganization of LHCII. The kinetics of this large-scale structural effect does not correspond to the light-induced fluorescence quenching, in contrast to the kinetics of structural changes in LHCII observable at low oxygen concentrations. Photo-conversion of 5% of the pool of all-trans violaxanthin to 9-cis isomer was observed under such conditions. Possible involvement of the violaxanthin isomerization in the process of structural rearrangements and excitation quenching in LHCII is discussed.
Keywords: LHCII; Violaxanthin; Carotenoid isomer; Fluorescence quenching;

Activation of the plant mitochondrial alternative oxidase: insights from site-directed mutagenesis by Ann L. Umbach; Miquel A. Gonzàlez-Meler; Charles R. Sweet; James N. Siedow (118-128).
The homodimeric cyanide-resistant alternative oxidase of plant mitochondria reduces oxygen to water without forming ATP. Arabidopsis thaliana alternative oxidase AOX1a is stimulated by pyruvate or other α-keto acids associating with a regulatory cysteine at position 78, by succinate in a serine-78 mutant, and by site-directed mutation of position 78 to glutamate. The mechanism of activation was explored with additional amino acid substitutions made at Cys-78 in AOX1a, which was functionally expressed in Escherichia coli. Oxidases with positively charged substitutions (Lys and Arg) were insensitive to pyruvate or succinate but were more active than the wild type without pyruvate. Uncharged substitutions (Gln, Leu) produced an inactive enzyme. These results indicate that activation may be due to conformational changes caused by charge repulsion between the dimer subunits and not through a direct role of α-keto acids in catalysis. Oxygen isotope fractionation experiments suggest that the charge of the amino acid at position 78 also affects the entry of oxygen into the active site. Therefore, the N-terminal portion of the protein containing residue 78 can indirectly affect both catalysis at the diiron active site and the path of oxygen to that site. In addition, both positively and negatively substituted alternative oxidases were stimulated by glyoxylate, suggesting the presence of a second activation site, possibly Cys-128.
Keywords: Alternative oxidase; Regulatory cysteine; α-Keto acid; Activity regulation; Plant mitochondria; Cyanide-resistant respiration;

New parameters reducing the interindividual variability of metabolic changes during muscle contraction in humans by Jean-Pierre Mattei; Geneviève Kozak-Ribbens; Magali Roussel; Yann Le Fur; Patrick J Cozzone; David Bendahan (129-136).
Interindividual variations in skeletal muscle metabolism make comparative analyses difficult. In this study, we have addressed the issue of capturing the variability of metabolic performance observed during muscle exercise in humans by using an original method of normalization.Metabolic changes induced by various kinds of exercise were investigated using 31P magnetic resonance spectroscopy (MRS) at 4.7 T in 65 normal subjects (23 women and 42 men) and 12 patients with biopsy-proven muscular disorders.Large variations in the extent of PCr breakdown and intracellular acidosis were recorded among subjects and exercise protocols. For all the data pooled, the amplitude of mechanical performance accounts for 50% of these variations. When scaled to the work output, variations of PCr consumption account for 65% of pH changes through a linear relationship. This linear relationship was substantially improved (90%) when both variables were scaled to the square of work output performed (P1 and P2). By capturing most of the initial interindividual variability (90%), P1 vs. P2 relationship represents an ideal standardization procedure, independent of any anthropometric measurements. This relationship also discloses a significant link between the extent of PCr breakdown and intracellular acidosis regardless of exercise protocol. Moreover, changes in the slope of the P1 vs. P2 regression curve, as measured in old subjects and in selected patients, directly reflect alterations of energy production in muscle.
Keywords: Human muscle; Muscle physiology; Energy metabolism; 31P magnetic resonance spectroscopy; Muscular disease;