BBA - Bioenergetics (v.1607, #2-3)

Two conserved charged amino acids, aspartate-186 and arginine-190, localized in the aqueous head region of the iron–sulfur protein of the cytochrome bc 1 complex of yeast mitochondria, were mutated to alanine, glutamate, or asparagine and isoleucine, respectively. The R190I mutation resulted in the complete loss of antimycin- and myxothiazol-sensitive cytochrome c reductase activity due to loss of more than 60% of the iron–sulfur protein in the complex. Mitochondria isolated from the D186A mutant had a 50% decrease in cytochrome c reductase activity but no loss of the iron–sulfur protein or the [2Fe–2S] cluster. The midpoint potential of the [2Fe–2S] cluster of the D186A mutant was decreased from 281 to 178 mV. The D186E and D186N mutations did not result in a loss of cytochrome c reductase activity or content of iron–sulfur protein; however, the redox potential of the [2Fe–2S] cluster of D186N was decreased from 281 to 241 mV. Molecular modeling/dynamics studies predicted that substituting an alanine for Asp-186 causes global structural changes in the head group of the iron–sulfur protein resulting in changes in the orientation of the [2Fe–2S] cluster and consequently a lowered redox potential. The rate of electrogenic proton pumping in the bc 1 complex isolated from mutant D186A reconstituted into proteoliposomes decreased 64%; however, the H+/2e ratio of 1.9 was identical in the mutant and the wild-type complexes. The carboxyl binding reagent, N-(ethoxycarbonyl)-2-ethoxyl-1,2-dihydroquinoline (EEDQ) blocked electrogenic proton pumping in the bc 1 complex reconstituted into proteoliposomes without affecting electron transfer resulting in a decrease in the H+/2e ratio to 1.2 and 1.1, respectively. EEDQ was bound to the iron–sulfur protein and core protein II in both the wild type and the D186A mutant, indicating that Asp-186 of the iron–sulfur protein is not required for proton translocation in the bc 1 complex.
Keywords: Cytochrome bc 1 complex; Iron–sulfur protein; Proton pumping; Ubiquinol:cytochrome c reductase; Complex III; Mitochondrial electron transport chain; Yeast mitochondria;

The mitochondrial and prokaryotic proton-translocating NADH:ubiquinone oxidoreductases: similarities and dissimilarities of the quinone-junction sites by Vera G. Grivennikova; Robert Roth; Natalia V. Zakharova; Cecilia Hägerhäll; Andrei D. Vinogradov (79-90).
The catalytic properties of the rotenone-sensitive NADH:ubiquinone reductase (Complex I) in bovine heart submitochondrial particles and in inside-out vesicles derived from Paracoccus denitrificans and Rhodobacter capsulatus were compared. The prokaryotic enzymes catalyze the NADH oxidase and NADH:quinone reductase reactions with similar kinetic parameters as those for the mammalian Complex I, except for lower apparent affinities for the substrates—nucleotides. Unidirectional competitive inhibition of NADH oxidation by ADP-ribose, previously discovered for submitochondrial particles, was also evident for tightly coupled P. denitrificans vesicles, thus suggesting that a second, NAD+-specific site is present in the simpler prokaryotic enzyme. The inhibitor sensitivity of the forward and reverse electron transfer reactions was compared. In P. denitrificans and Bos taurus vesicles different sensitivities to rotenone and Triton X-100 for the forward and reverse electron transfer reactions were found. In bovine heart preparations, both reactions showed the same sensitivity to piericidin, and the inhibition was titrated as a straight line. In P. denitrificans, the forward and reverse reactions show different sensitivity to piericidin and the titrations of both activities were curvilinear with apparent I 50 (expressed as mole of inhibitor per mole of enzyme) independent of the enzyme concentration. This behavior is explained by a model involving two different sites rapidly interacting with piericidin within the hydrophobic phase.
Keywords: NADH:ubiquinone reductase; Complex I; NDH-1; Ubiquinone; Piericidin; Tightly bound inhibitor; Respiratory chain;

Addition of N,N,N′,N′-tetramethyl-p-phenylendiamine (TMPD) to thylakoid membranes isolated from pea leaves initiates the appearance of peak I in the polyphasic rise of chlorophyll (Chl) fluorescence observed during strong illumination, making it similar to that observed in leaves or intact chloroplasts. This effect depends on TMPD concentration and incubation period of isolated thylakoids with TMPD. The resolution of I-peak in the presence of weak concentrations of TMPD which reduced the overlap between I- and P-peaks, resulted from a decreased reduction of both fast and slow plastoquinone (PQ) pools of the granal and stromal thylakoids, respectively, as TMPD effectively accepts electrons from reduced PQ. High concentrations of TMPD markedly decreased the J–I–P phase of fluorescence rise and greatly retarded the I–P step rise. Accumulation of oxidized TMPD in the thylakoid lumen accelerated the re-oxidation of the acceptor side of Photosystem II (PSII) as illustrated by a two-fold increase in the magnitude of the fast component and complete suppression of the middle component of the variable Chl fluorescence (F v) decay in the dark. Evidently, exogenous addition of high concentrations of TMPD prevented the light-induced reduction of the slow PQ pool.
Keywords: Chlorophyll fluorescence; Photosystem II; TMPD; Thylakoid;

The diffusion of plastoquinol in the chloroplast thylakoid membrane is modelled using Monte Carlo techniques. The integral proteins are seen as obstacles to diffusion, and features of percolation theory emerge. Thus, the diffusion coefficient diminishes with increasing distance and there is a critical threshold of protein concentration, above which the long-range diffusion coefficient is zero. The area occupied by proteins in vivo is assessed and appears to be around this threshold, as determined from calculations assuming randomly distributed noninteracting proteins. Slight changes in the protein arrangement lead to pronounced changes in diffusion behaviour under such conditions. Mobility of the proteins increases the protein occupancy threshold, while boundary lipids impermeable to PQ diffusion decrease it. Further, the obstruction of plastoquinone/plastoquinol binding sites in a random arrangement is evaluated.
Keywords: Thylakoid membrane; Plastoquinone; Diffusion; Percolation; Microdomain; Monte Carlo simulation;

Concentrations of adenylate species and free magnesium (Mg2+) within cells are mediated by the equilibrium governed by adenylate kinase (AK), the enzyme abundant in plants in chloroplast stroma and intermembrane spaces of chloroplasts and mitochondria. Ratios of free and Mg-bound adenylates (linked to the values of [Mg2+] established under AK equilibrium) can be rationalized in terms of the overall dependence of concentrations of Mg2+ and free and Mg-bound adenylates, as well as electric potential values across the inner membranes of mitochondria and chloroplasts. The potential across the inner mitochondrial membrane, by driving adenylate translocators, equilibrates free adenylates across the inner membrane according to the Nernst equation and contributes to the ATPtotal/ADPtotal ratio in the cytosol. The ratio affects the exchange of free adenylates with chloroplasts and this, in turn, influences the value of potential across the inner chloroplast membrane. From measurements of subcellular ATPtotal/ADPtotal ratios, we suggest a method of estimating the values of potential across inner membranes of mitochondria and chloroplasts in vivo, which allows a comparison of the operation of these organelles under different physiological conditions. We discuss also how the equilibration of adenylates by AK drives adenylate transport across membranes, and establishes [Mg2+] in the cytosol and chloroplast stroma, maintaining the rates of photosynthesis and respiration. This provides a tool for metabolomic research, by which the determined concentrations of adenylate species could be used for computation of essential metabolic parameters in the cell and in subcellular compartments.
Keywords: Adenylate kinase; Chloroplast; Free magnesium; Membrane potential; Metabolomics; Mitochondrion;

The state transitions of the cyanobacterium Synechococcus sp. PCC 7002 and of three mutant strains, which were impaired in PsaE-dependent cyclic electron transport (psaE ), respiratory electron transport (ndhF ) and both activities (psaE ndhF ), were analyzed. Dark incubation of the wild type and psaE cells led to a transition to state 2, while the ndhF strains remained in state 1 after dark incubation. The ndhF cells adapted to state 2 when the cells were incubated under anaerobic conditions or in the presence of potassium cyanide; these results suggest that the ndhF cells were inefficient in performing state 1 to state 2 transitions in the dark unless cytochrome oxidase activity was inhibited. In the state 2 to state 1 transition of wild-type cells induced by light in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), there was still a significant reduction of the interphotosystem electron carriers by both respiration and cyclic electron flow around PSI. Kinetic analysis of the state 2 to state 1 transition shows that, in the absence of PSII activity, the relative contribution to the reduced state of the interphotosystem electron carriers by respiratory and cyclic electron transfer is about 72% and 28%, respectively. The state 2 to state 1 transition was prevented by the cytochrome b 6 f inhibitor 2,5-dibromo-3-methyl-6-isopropylbenzoquinone (DBMIB). On the other hand, the state 1 to state 2 transition was induced by DBMIB with half times of approximately 8 s in all strains. The externally added electron acceptor 2,5-dimethyl-benzoquinone (DMBQ) induced a state 2 to state 1 transition in the dark and this transition could be prevented by DBMIB. The light-induced oxidation of P700 showed that approximately 50% of PSI could be excited by 630-nm light absorbed by phycobilisomes (PBS) under state 2 conditions. P700 oxidation measurements with light absorbed by PBS also showed that the dark-induced state 1 to state 2 transition occurred in wild-type cells but not in the ndhF cells. The possible mechanism for sensing an imbalanced light regime in cyanobacterial state transitions is discussed.
Keywords: Synechococcus PCC 7002; Electron transport; Mutant;

Dissecting a cyanobacterial proteolytic system: efficiency in inducing degradation of the D1 protein of photosystem II in cyanobacteria and plants by Eira Kanervo; Cornelia Spetea; Yoshitaka Nishiyama; Norio Murata; Bertil Andersson; Eva-Mari Aro (131-140).
A chromatography fraction, prepared from isolated thylakoids of a fatty acid desaturation mutant (Fad6/desA∷Kmr) of the cyanobacterium Synechocystis 6803, could induce an initial cleavage of the D1 protein in Photosystem II (PSII) particles of Synechocystis 6803 mutant and Synechococcus 7002 wild type as well as in supercomplexes of PSII-light harvesting complex II of spinach. Proteolysis was demonstrated both in darkness and in light as a reduction in the amount of full-length D1 protein or as a production of C-terminal initial degradation fragments. In the Synechocystis mutant, the main degradation fragment was a 10-kDa C-terminal one, indicating an initial cleavage occurring in the cytoplasmic DE-loop of the D1 protein. A protein component of 70–90 kDa isolated from the chromatographic fraction was found to be involved in the production of this 10-kDa fragment. In spinach, only traces of the corresponding fragment were detected, whereas a 24-kDa C-terminal fragment accumulated, indicating an initial cleavage in the lumenal AB-loop of the D1 protein. Also in Synechocystis the 24-kDa fragment was detected as a faint band. An antibody raised against the Arabidopsis DegP2 protease recognized a 35-kDa band in the proteolytically active chromatographic fraction, suggesting the existence of a lumenal protease that may be the homologue DegP of Synechocystis. The identity of the other protease cleaving the D1 protein in the DE-loop exposed on the stromal (cytoplasmic) side of the membrane is discussed.
Keywords: Cyanobacterium; D1 protein; DegP; FtsH; Photosystem II; Synechocystis 6803;

The membrane-bound proton pumping inorganic pyrophosphate synthase/pyrophosphatase (H+-PPi synthase/H+-PPase) from the photosynthetic bacterium Rhodospirillum rubrum was functionally expressed in Escherichia coli C43(DE3) cells. Based on a new topology model of the enzyme, charged residues predicted to be located near or within the membrane were selected for site-directed mutagenesis. Several of these mutations resulted in an almost complete inactivation of the enzyme. Four mutated residues appear to show a selective impairment of proton translocation and are thus likely to be involved in coupling pyrophosphate hydrolysis with electrogenic proton pumping. Two of these mutations, R176K and E584D, caused increased tolerance to salt. In addition, the former mutation caused an increased K m of one order of magnitude for the hydrolysis reaction. These results and their possible implications for the enzyme function are discussed.
Keywords: Inorganic pyrophosphate; Pyrophosphatase; Inorganic pyrophosphate synthase; Proton pumping pyrophosphatase; Phosphohydrolase; Rhodospirillum rubrum;

The dependence of algal H2 production on Photosystem II and O2 consumption activities in sulfur-deprived Chlamydomonas reinhardtii cells by T.K. Antal; T.E. Krendeleva; T.V. Laurinavichene; V.V. Makarova; M.L. Ghirardi; A.B. Rubin; A.A. Tsygankov; M. Seibert (153-160).
Chlamydomonas reinhardtii cultures, deprived of inorganic sulfur, undergo dramatic changes during adaptation to the nutrient stress [Biotechnol. Bioeng. 78 (2002) 731]. When the capacity for Photosystem II (PSII) O2 evolution decreases below that of respiration, the culture becomes anaerobic [Plant Physiol. 122 (2000) 127]. We demonstrate that (a) the photochemical activity of PSII, monitored by in situ fluorescence, also decreases slowly during the aerobic period; (b) at the exact time of anaerobiosis, the remaining PSII activity is rapidly down regulated; and (c) electron transfer from PSII to PSI abruptly decreases at that point. Shortly thereafter, the PSII photochemical activity is partially restored, and H2 production starts. Hydrogen production, which lasts for 3–4 days, is catalyzed by an anaerobically induced, reversible hydrogenase. While most of the reductants used directly for H2 gas photoproduction come from water, the remaining electrons must come from endogenous substrate degradation through the NAD(P)H plastoquinone (PQ) oxido-reductase pathway. We propose that the induced hydrogenase activity provides a sink for electrons in the absence of other alternative pathways, and its operation allows the partial oxidation of intermediate photosynthetic carriers, including the PQ pool, between PSII and PSI. We conclude that the reduced state of this pool, which controls PSII photochemical activity, is one of the main factors regulating H2 production under sulfur-deprived conditions. Residual O2 evolved under these conditions is probably consumed mostly by the aerobic oxidation of storage products linked to mitochondrial respiratory processes involving both the cytochrome oxidase and the alternative oxidase. These functions maintain the intracellular anaerobic conditions required to keep the hydrogenase enzyme in the active, induced form.
Keywords: Chlamydomonas reinhardtii; Photosystem II; O2 consumption;

Interaction between F1-ATPase activity stimulating oxyanions and noncatalytic sites of coupling factor CF1 was studied. Carbonate, borate and sulfite anions were shown to inhibit tight binding of [14C]ATP and [14C]ADP to CF1 noncatalytic sites. The demonstrated change of their inhibitory efficiency in carbonate–borate–sulfite order coincides with the previously found change in efficiency of these anions as stimulators of CF1-ATPase activity [Biochemistry (Mosc.) 43 (1978) 1206–1211]. Inhibition of tight nucleotide binding to noncatalytic sites was accompanied by stimulation of nucleotide binding to catalytic sites. This suggests that stimulation of CF1-ATPase activity is caused by interaction between oxyanions and noncatalytic sites. A most efficient stimulator of CF1-ATPase activity, sulfite oxyanion, appeared to be a competitive inhibitor with respect to ATP and a partial noncompetitive inhibitor with respect to ADP. The inhibition weakened with increasing time of CF1 incubation with sulfite and nucleotides. Sulfite is believed to inhibit fast reversible interaction between nucleotides and noncatalytic sites and to produce no effect on subsequent tight binding of nucleotides. A possible mechanism of the oxyanion-stimulating effect is discussed.
Keywords: Chloroplast coupling factor 1 (CF1); F1-ATPase; Noncatalytic site; Oxyanion;

FRET reveals changes in the F1–stator stalk interaction during activity of F1F0-ATP synthase by Paul D Gavin; Rodney J Devenish; Mark Prescott (167-179).
A stator is proposed as necessary to prevent futile rotation of the F1 catalytic sector of mitochondrial ATP synthase (mtATPase) during periods of ATP synthesis or ATP hydrolysis. Although the second stalk of mtATPase is generally believed to fulfil the role of a stator capable of withstanding the stress produced by rotation of the central rotor, there is little evidence to directly support this view. We show that interaction between two candidate proteins of the second stalk, OSCP and subunit b, fused at their C-termini to GFP variants and assembled into functional mtATPase can be monitored in mitochondria using fluorescence resonance energy transfer (FRET). Substitution of native OSCP with a variant containing a glycine 166 to asparagine (G166N) substitution yielded a metastable complex. In contrast to the enzyme containing native OSCP, FRET could be irreversibly lowered for the enzyme containing G166N at a rate that correlated closely with the rate of enzyme activity (ATP hydrolysis). The non-hydrolysable ATP analogue, AMP-PCP did not have this effect. We conclude that two candidate proteins of the stator stalk, OSCP and b, are subject to stresses during enzyme catalytic activity commensurate with their role as a part of a stator stalk.
Keywords: ATP synthase; Stator stalk; Fluorescent protein; GFP; FRET;

Each yeast mitochondrial F1F0-ATP synthase complex contains a single copy of subunit 8 by Andrew N. Stephens; Phillip Nagley; Rodney J. Devenish (181-189).
The stoichiometry of subunit 8 in yeast mitochondrial F1F0-ATP synthase (mtATPase) has been evaluated using an immunoprecipitation approach. Single HA or FLAG epitopes were introduced at the N-terminus of subunit 8. Expression of each tagged subunit 8 variant in yeast cells lacking endogenous subunit 8 restored a respiratory phenotype and had little measurable effect on ATP hydrolase activity of the isolated enzyme. Moreover, the two epitope-tagged subunit 8 variants could be stably co-expressed in the same host cells and both of HA-Y8 and FLAG-Y8 could be detected in ATP synthase complexes isolated by native gel electrophoresis. Mitochondria isolated from each yeast strain were solubilized to release ATP synthase complexes in either the monomeric or dimeric forms. In each case, monoclonal antibodies directed against either the FLAG or HA epitope could immunoprecipitate intact ATP synthase complexes. When both HA-Y8 and FLAG-Y8 were co-expressed in cells, monomeric ATP synthases contained only a single subunit 8 variant after immunoprecipitation, corresponding to the particular antibody used (HA or FLAG). By contrast, both subunit 8 variants were recovered in samples of immunoprecipitated dimeric ATP synthase complexes, irrespective of the antibody used. We conclude that each monomeric yeast mitochondrial ATP synthase complex contains a single copy of subunit 8.
Keywords: F1F0-ATP synthase; Proton pore; Stator stalk; Stoichiometry; Subunit 8; Yeast mitochondria;

Monitoring cytochrome redox changes in the mitochondria of intact cells using multi-wavelength visible light spectroscopy by Veronica S. Hollis; Miriam Palacios-Callender; Roger J. Springett; David T. Delpy; Salvador Moncada (191-202).
We have developed an optical system based on visible light spectroscopy for the continuous study of changes in the redox states of mitochondrial cytochromes in intact mammalian cells. Cells are suspended in a closed incubation chamber in which oxygen and nitric oxide (NO) concentrations can be monitored during respiration. Simultaneously the cells are illuminated with a broad-band tungsten–halogen light source. Emergent light in the visible region (from 490–650 nm) is detected using a spectrophotometer and charge-coupled device camera system. Intensity spectra are then converted into changes in optical attenuation from a ‘steady-state’ baseline. The oxidised-minus-reduced absorption spectra of the mitochondrial cytochromes are fitted to the attenuation spectra using a multi-wavelength least-squares algorithm. Thus, the system can measure changes in the redox states of the cytochromes during cellular respiration. Here we describe this novel methodology and demonstrate its validity by monitoring the action of known respiratory chain inhibitors, including the endogenous signalling molecule NO, on cytochrome redox states in human leukocytes.
Keywords: Visible light spectroscopy; Cellular respiration; Cytochrome redox state; Mitochondrial inhibitor; Nitric oxide;

The crystal structure of the spinach plastocyanin double mutant G8D/L12E gives insight into its low reactivity towards photosystem 1 and cytochrome f by Hanna Jansson; Mats Ökvist; Frida Jacobson; Mikael Ejdebäck; Örjan Hansson; Lennart Sjölin (203-210).
Plastocyanin (Pc) is a copper-containing protein, which functions as an electron carrier between the cytochrome b 6 f and photosystem 1 (PS1) complexes in the photosynthetic electron transfer (ET) chain. The ET is mediated by His87 situated in the hydrophobic surface in the north region of Pc. Also situated in this region is Leu12, which mutated to other amino acids severely disturbs the ET from cytochrome f and to PS1, indicating the importance of the hydrophobic surface. The crystal structure of the Pc double mutant G8D/L12E has been determined to 2.0 Å resolution, with a crystallographic R-factor of 18.3% (R free=23.2%). A comparison with the wild-type structure reveals that structural differences are limited to the sites of the mutations. In particular, there is a small but significant change in the hydrophobic surface close to His87. Evidently, this leads to a mismatch in the reactive complex with the redox partners. For PS1 this results in a 20 times weaker binding and an eightfold slower ET as determined by kinetic measurements. The mutations that have been introduced do not affect the optical absorption spectrum. However, there is a small change in the EPR spectrum, which can be related to changes in the copper coordination geometry.
Keywords: Crystal structure; Electron transfer; Hydrophobic patch; Kinetics; Plastocyanin; Protein-protein interaction;

Cytoplasmic pH regulation mediated by the H+-ATPase was examined with the aid of computer simulation. The data obtained with our simulation model were consistent with the experimental data and the simulation clarified the following points that may be difficult to be clarified with experimental studies. (1) The change in the enzyme amount controlled by cytoplasmic pH was essential for the pH regulation. (2) No significant change in internal pH was observed in acidic surroundings even if the proton transport activity of the H+-ATPase changed greater than sixfold. (3) The cytoplasmic pH homeostasis can be maintained even when the biosynthetic rate of the enzyme decreased by 50%. These results suggested that this regulatory system has an ability to maintain the pHin homeostasis even under harsh conditions that decrease cellular metabolic activities.
Keywords: pH regulation; Computer simulation; H+-ATPase;