BBA - Bioenergetics (v.1504, #2-3)
Evidence for a quinone binding site close to the interface between NUOD and NUOB subunits of Complex I by Isabelle Prieur; Joël Lunardi; Alain Dupuis (173-178).
Piericidin, rotenone and pyridaben are specific inhibitors of the NADH–ubiquinone oxidoreductase (Complex I) that bind to its ubiquinone binding site(s). Using site directed mutagenesis, we demonstrate that residues G409, D412, R413 and V407 of the C-terminus of Complex I NUOD subunit are directly involved in the binding of these inhibitors. We propose that the corresponding inhibitor/quinone binding site would be located close to NUOD–NUOB interface.
Keywords: Complex I; Mitochondria; Piericidin; Rotenone; Ubiquinone; (Rhodobacter capsulatus);
Fungal respiration: a fusion of standard and alternative components by Tim Joseph-Horne; Derek W. Hollomon; Paul M. Wood (179-195).
In animals, electron transfer from NADH to molecular oxygen proceeds via large respiratory complexes in a linear respiratory chain. In contrast, most fungi utilise branched respiratory chains. These consist of alternative NADH dehydrogenases, which catalyse rotenone insensitive oxidation of matrix NADH or enable cytoplasmic NADH to be used directly. Many also contain an alternative oxidase that probably accepts electrons directly from ubiquinol. A few fungi lack Complex I. Although the alternative components are non-energy conserving, their organisation within the fungal electron transfer chain ensures that the transfer of electrons from NADH to molecular oxygen is generally coupled to proton translocation through at least one site. The alternative oxidase enables respiration to continue in the presence of inhibitors for ubiquinol:cytochrome c oxidoreductase and cytochrome c oxidase. This may be particularly important for fungal pathogens, since host defence mechanisms often involve nitric oxide, which, whilst being a potent inhibitor of cytochrome c oxidase, has no inhibitory effect on alternative oxidase. Alternative NADH dehydrogenases may avoid the active oxygen production associated with Complex I. The expression and activity regulation of alternative components responds to factors ranging from oxidative stress to the stage of fungal development.
Keywords: Mitochondria; Fungus; Mitochondrial electron transport; NAD(P)H:ubiquinone oxidoreductase; Alternative oxidase; Protonmotive force;
Generation and propagation of radical reactions on proteins by Clare L. Hawkins; Michael J. Davies (196-219).
The oxidation of proteins by free radicals is thought to play a major role in many oxidative processes within cells and is implicated in a number of human diseases as well as ageing. This review summarises information on the formation of radicals on peptides and proteins and how radical damage may be propagated and transferred within protein structures. The emphasis of this article is primarily on the deleterious actions of radicals generated on proteins, and their mechanisms of action, rather than on enzymatic systems where radicals are deliberately formed as transient intermediates. The final section of this review examines the control of protein oxidation and how such damage might be limited by antioxidants.
Keywords: Protein oxidation; Amino acid oxidation; Free radical; Peroxyl radical; Alkoxyl radical; Nitrogen-centered radical; Carbon-centered radical;
Stoichiometry of subunit e in rat liver mitochondrial H+-ATP synthase and membrane topology of its putative Ca2+-dependent regulatory region by Naokatu Arakaki; Yumiko Ueyama; Mayumi Hirose; Toshiki Himeda; Hirofumi Shibata; Shiroh Futaki; Kouki Kitagawa; Tomihiko Higuti (220-228).
Previous studies have revealed that residues 34–65 of subunit e of mitochondrial H+-ATP synthase are homologous with the Ca2+-dependent tropomysin-binding region for troponin T and have suggested that subunit e could be involved in the Ca2+-dependent regulation of H+-ATP synthase activity. In this study, we determined the content of subunit e in H+-ATP synthase purified from rat liver mitochondria, and we also investigated the membrane topology of a putative Ca2+-dependent regulatory region of subunit e using an antibody against peptide corresponding to residues 34–65 of subunit e. Quantitative immunoblot analysis of subunit e in the purified H+-ATP synthase revealed that 1 mol of H+-ATP synthase contained 2 mol of subunit e. The ATPase activity of mitoplasts, in which the C-side of F0 is present on the outer surface of the inner membrane, was significantly stimulated by the addition of the antibody, while the ATPase activity of submitochondrial particles and purified H+-ATP synthase was not stimulated. The antibody bound to mitoplasts but not to submitochondrial particles. These results suggest that the putative Ca2+-dependent regulatory region of subunit e is exposed on the surface of the C-side of F0 and that subunit e is involved in the regulation of mitochondrial H+-ATP synthase activity probably via its putative Ca2+-dependent regulatory region.
Keywords: Mitochondria; H+-ATP synthase; Subunit e; Stoichiometry; Membrane topology;
Temperature-induced decoupling of phycobilisomes from reaction centers by Ye Li; Jianping Zhang; Jie Xie; Jingquan Zhao; Lijin Jiang (229-234).
Temperature-induced decoupling of phycobilisomes (PBSs) from the reaction centers in the PBS-thylakoid membrane complexes was observed at 0°C. The fluorescence yields of photosystem (PS) I and PSII decreased and that of PBSs increased with selective excitation of PBSs at 0°C, while the yield of PBSs decreased and those of the two photosystems increased with selective excitation of chlorophyll a at room temperature (RT). It indicated that the decoupling of PBSs from the two photosystems led to changes of energy transfer efficiencies, which can be explained by partial detachment of PBSs from thylakoid membrane. The temperature-dependent processes were reversible, i.e. with temperature going up to RT, the complexes could restore to the functionally coupled state with a time constant about 30 s. Based on these results, it could be deduced that PBSs should be in parallel connection with the two photosystems.
Keywords: Phycobilisome-thylakoid membrane complex; Photosystem; Energy transfer; Decoupling;
Modification of inhibitor binding sites in the cytochrome bf complex by directed mutagenesis of cytochrome b 6 in Synechococcus sp. PCC 7002 by Tou-Xang Lee; Sabine U Metzger; Yoon Shin Cho; John Whitmarsh; Toivo Kallas (235-247).
The cytochrome bf complex, which links electron transfer from photosystem II to photosystem I in oxygenic photosynthesis, has not been amenable to site-directed mutagenesis in cyanobacteria. Using the cyanobacterium Synechococcus sp. PCC 7002, we have successfully modified the cytochrome b 6 subunit of the cytochrome bf complex. Single amino acid substitutions in cytochrome b 6 at the positions D148, A154, and S159 revealed altered binding of the quinol-oxidation inhibitors 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), myxothiazol, and stigmatellin. Cytochrome bf and mitochondrial-type cytochrome bc 1 complexes are closely related in structure and function but exhibit quite different inhibitor specificities. Cytochrome bf complexes are insensitive to myxothiazol and sensitive to DBMIB, whereas cytochrome bc 1 complexes are sensitive to myxothiazol and relatively insensitive to DBMIB. Measurements of flash-induced and steady-state electron transfer rates through the cytochrome bf complex revealed increased resistance to DBMIB in the mutants A154G and S159A, increased resistance to stigmatellin in A154G, and created sensitivity to myxothiazol in the mutant D148G. Therefore these mutations made the cytochrome bf complex more like the cytochrome bc 1 complex. This work demonstrates that cyanobacteria can be used as effective models to investigate structure–function relationships in the cytochrome bf complex.
Keywords: Photosynthesis; Cytochrome bf complex; Quinol-oxidation; Inhibitor site; Directed mutagenesis; Cyanobacteria;
The rapid mode of calcium uptake into heart mitochondria (RaM): comparison to RaM in liver mitochondria by Linas Buntinas; Karlene K Gunter; Genevieve C Sparagna; Thomas E Gunter (248-261).
A mechanism of Ca2+ uptake, capable of sequestering significant amounts of Ca2+ from cytosolic Ca2+ pulses, has previously been identified in liver mitochondria. This mechanism, the Rapid Mode of Ca2+ uptake (RaM), was shown to sequester Ca2+ very rapidly at the beginning of each pulse in a sequence [Sparagna et al. (1995) J. Biol. Chem. 270, 27510–27515]. The existence and properties of RaM in heart mitochondria, however, are unknown and are the basis for this study. We show that RaM functions in heart mitochondria with some of the characteristics of RaM in liver, but its activation and inhibition are quite different. It is feasible that these differences represent different physiological adaptations in these two tissues. In both tissues, RaM is highly conductive at the beginning of a Ca2+ pulse, but is inhibited by the rising [Ca2+] of the pulse itself. In heart mitochondria, the time required at low [Ca2+] to reestablish high Ca2+ conductivity via RaM i.e. the ‘resetting time’ of RaM is much longer than in liver. RaM in liver mitochondria is strongly activated by spermine, activated by ATP or GTP and unaffected by ADP and AMP. In heart, RaM is activated much less strongly by spermine and unaffected by ATP or GTP. RaM in heart is strongly inhibited by AMP and has a biphasic response to ADP; it is activated at low concentrations and inhibited at high concentrations. Finally, an hypothesis consistent with the data and characteristics of liver and heart is presented to explain how RaM may function to control the rate of oxidative phosphorylation in each tissue. Under this hypothesis, RaM functions to create a brief, high free Ca2+ concentration inside mitochondria which may activate intramitochondrial metabolic reactions with relatively small amounts of Ca2+ uptake. This hypothesis is consistent with the view that intramitochondrial [Ca2+] may be used to control the rate of ADP phosphorylation in such a way as to minimize the probability of activating the Ca2+-induced mitochondrial membrane permeability transition (MPT).
Keywords: Mitochondria; Heart mitochondria; Calcium uptake; Calcium pulse; Calcium signaling; Metabolism; Permeability transition; Apoptosis;
The oxidation state of the photosystem II manganese cluster influences the structure of manganese stabilizing protein by S.K Hong; S.A Pawlikowski; K.A Vander Meulen; C.F Yocum (262-274).
Exposure of photosystem II membranes to trypsin that has been treated to inhibit chymotrypsin activity produces limited hydrolysis of manganese stabilizing protein. Exposure to chymotrypsin under the same conditions yields substantial digestion of the protein. Further probing of the unusual insensitivity of manganese stabilizing protein to trypsin hydrolysis reveals that increasing the temperature from 4 to 25°C will cause some acceleration in the rate of proteolysis. However, addition of low (100 μM) concentrations of NH2OH, that are sufficient to reduce, but not destroy, the photosystem II Mn cluster, causes a change in PS II-bound manganese stabilizing protein that causes it to be rapidly digested by trypsin. Immunoblot analyses with polyclonal antibodies directed against the N-terminus of the protein, or against the entire sequence show that trypsin cleavage produces two distinct peptide fragments estimated to be in the 17–20 kDa range, consistent with proposals that there are 2 mol of the protein/mol photosystem II. The correlation of trypsin sensitivity with Mn redox state(s) in photosystem II suggest that manganese stabilizing protein may interact either directly with Mn, or alternatively, that the polypeptide is bound to another protein of the photosystem II reaction center that is intimately involved in binding and redox activity of Mn.
Keywords: Manganese stabilizing protein; Photosystem II; Trypsin; Manganese; Oxygen evolution;
Chloroplastic ascorbate peroxidase is the primary target of methylviologen-induced photooxidative stress in spinach leaves: its relevance to monodehydroascorbate radical detected with in vivo ESR by Jun’ichi Mano; Chiaki Ohno; Yoshinori Domae; Kozi Asada (275-287).
Methylviologen (MV) induces oxidative damages in leaves. In order to understand its mechanism we studied initial biochemical events under light in MV-fed spinach leaves. When isolated chloroplasts were illuminated in the presence of MV, both stromal and thylakoid-bound ascorbate peroxidases (APX) were inactivated rapidly at the same rates, and their inactivation was retarded by ascorbate (AsA) at higher concentrations. Since MV accelerates the photoproduction of O2 − in Photosystem (PS) I and simultaneously inhibits the photoreduction of monodehydroascorbate (MDA) to AsA, the inactivation of APX was attributed to the loss of AsA and accumulation of H2O2 in the stroma. Following APX, superoxide dismutase and NADP+-glyceraldehyde 3-phosphate dehydrogenase, both of which are vulnerable to H2O2, were inactivated by MV plus light. Dehydroascorbate reductase, monodehydroascorbate reductase, PS II, PS I and ferredoxin-NADP+ reductase were far less sensitive to the treatment. In the treated leaves, cytosolic APX and guaiacol-specific peroxidase were also inactivated, but slower than chloroplastic APXs were. Catalase was not inactivated. Thus the MV-induced photooxidative damages of leaves are initiated with the inactivation of chloroplastic APXs and develop via the inactivation of other H2O2-sensitive targets. The decay half-life of the MDA signal after a short illumination in the leaves, as determined by in vivo electron spin resonance spectrometry (ESR), was prolonged when the H2O2-scavenging capacity of the leaf cells was abolished by the inactivation of chloroplastic and cytosolic APXs. The measurement of MDA in leaves by ESR, therefore, allows to estimate in vivo cellular capacity to scavenge the photoproduced H2O2.
Keywords: Ascorbate peroxidase; Chloroplast; Electron spin resonance; Monodehydroascorbate radical; Methylviologen; Oxidative stress;
Photoreducible high spin iron electron paramagnetic resonance signals in dark-adapted Photosystem II: are they oxidised non-haem iron formed from interaction of oxygen with PSII electron acceptors? by Jonathan H.A. Nugent (288-298).
An electron paramagnetic resonance (EPR) signal near g=6 in Photosystem II (PSII) membranes has been assigned to a high spin form of cytochrome (Cyt) b 559 (R. Fiege, U. Schreiber, G. Renger, W. Lubitz, V.A. Shuvalov, FEBS Lett. 377 (1995) 325–329). Here we have further investigated the origin of this signal. A slow formation of the signal during storage in the dark is observed in oxygen-evolving PSII membranes, which correlate with the oxidation of Fe2+ by plastosemiquinone or oxygen. Removal of oxygen inhibits formation of the high spin iron signal. The g=6 EPR signal is photoreduced at cryogenic temperatures and is restored slowly by subsequent dark storage at 77 K. The amplitude of the photoreduced signal increases as the pH is lowered, which shows that the origin is not the hydroxyl ligated Cyt b 559 species proposed previously. Different cryoprotectants also influence the amplitude and lineshape of the high spin iron signal in a manner suggesting that smaller cryoprotectants can penetrate the iron environment. A correlation between the high spin iron and g=1.6 EPR signal assigned to an interaction involving the semiquinones of Qa and Qb is shown. It is concluded that the appearance of the high spin iron signal in oxygen-evolving PSII membranes involves reduced PSII electron acceptors and oxygen and suggests that the signal is from the non-haem iron of PSII.
Keywords: Photosynthesis; Photosystem II; Plastoquinone; Non-heme iron; Cytochrome b 559;
Two functionally distinct manganese clusters formed by introducing a mutation in the carboxyl terminus of a photosystem II reaction center polypeptide, D1, of the green alga Chlamydomonas reinhardtii by Aya Hatano-Iwasaki; Jun Minagawa; Yorinao Inoue; Yuichiro Takahashi (299-310).
To study the function of the carboxyl-terminal domain of a photosystem II (PSII) reaction center polypeptide, D1, chloroplast mutants of the green alga Chlamydomonas reinhardtii have been generated in which Leu-343 and Ala-344 have been simultaneously or individually replaced by Phe and Ser, respectively. The mutants carrying these replacements individually, L343F and A344S, showed a wild-type phenotype. In contrast, the double mutant, L343FA344S, evolved O2 at only 20–30% of the wild-type rate and was unable to grow photosynthetically. In this mutant, PSII accumulated to 60% of the wild-type level, indicating that the O2-evolving activity per PSII was reduced to approximately half that of the wild-type. However, the amount of Mn atom detected in the thylakoids suggested that a normal amount of Mn cluster was assembled. An investigation of the kinetics of flash-induced fluorescence yield decay revealed that the electron transfer from Q− A to QB was not affected. When a back electron transfer from Q− A to a donor component was measured in the presence of 3-(3,4-dichlorophenol)-1,1-dimethylurea, a significantly slower component of the Q− A oxidation was detected in addition to the normal component that corresponds to the back electron transfer from the Q− A to the S2-state of the Mn cluster. Thermoluminescence measurements revealed that L343FA344S cells contained two functionally distinct Mn clusters. One was equivalent to that of the wild-type, while the other was incapable of water oxidation and was able to advance the transition from the S1-state to the S2-state. These results suggested that a fraction of the Mn cluster had been impaired by the L343FA344S mutation, leading to decreased O2 evolution. We concluded that the structure of the C-terminus of D1 is critical for the formation of the Mn cluster that is capable of water oxidation, in particular, transition to higher S-states.
Keywords: Photosystem II; O2 evolution; psbA; Manganese cluster; Chloroplast transformation; Chlamydomonas reinhardtii;
Long-lived charge-separated states in bacterial reaction centers isolated from Rhodobacter sphaeroides by Frank van Mourik; Michael Reus; Alfred R. Holzwarth (311-318).
We studied the accumulation of long-lived charge-separated states in reaction centers isolated from Rhodobacter sphaeroides, using continuous illumination, or trains of single-turnover flashes. We found that under both conditions a long-lived state was produced with a quantum yield of about 1%. This long-lived species resembles the normal P+Q− state in all respects, but has a lifetime of several minutes. Under continuous illumination the long-lived state can be accumulated, leading to close to full conversion of the reaction centers into this state. The lifetime of this accumulated state varies from a few minutes up to more than 20 min, and depends on the illumination history. Surprisingly, the lifetime and quantum yield do not depend on the presence of the secondary quinone, QB. Under oxygen-free conditions the accumulation was reversible, no changes in the normal recombination times were observed due to the intense illumination. The long-lived state is responsible for most of the dark adaptation and hysteresis effects observed in room temperature experiments. A simple method for quinone extraction and reconstitution was developed.
Keywords: Reaction center; Protein quake; Charge recombination; Protein conformation; Charge separation;
Evaluation of the hydrogen bonding interactions and their effects on the oxidation-reduction potentials for the riboflavin complex of the Desulfovibrio vulgaris flavodoxin by Fu-Chung Chang; Luke H Bradley; Richard P Swenson (319-328).
The oxidation-reduction potentials for the riboflavin complex of the Desulfovibrio vulgaris flavodoxin are substantially different from those of the flavin mononucleotide (FMN) containing native protein, with the midpoint potential for the semiquinone-hydroquinone couple for the riboflavin complex being 180 mV less negative. This increase has been attributed to the absence in the riboflavin complex of unfavorable electrostatic effects of the dianionic 5′-phosphate of the FMN on the stability of the flavin hydroquinone anion. In this study, 15N and 1H-15N heteronuclear single-quantum coherence nuclear magnetic resonance spectroscopic studies demonstrate that when bound to the flavodoxin, (1) the N(1) of the riboflavin hydroquinone remains anionic at pH 7.0 so the protonation of the hydroquinone is not responsible for this increase, (2) the N(5) position is much more exposed and may be hydrogen bonded to solvent, and (3) that while the hydrogen bonding interaction at the N(3)H appears stronger, that at the N(5)H in the reduced riboflavin is substantially weaker than for the native FMN complex. Thus, the higher reduction potential of the riboflavin complex is primarily the consequence of altered interactions with the flavin ring that affect hydrogen bonding with the N(5)H that disproportionately destabilize the semiquinone state of the riboflavin rather than through the absence of the electrostatic effects of the 5′-phosphate on the hydroquinone state.
Keywords: Flavodoxin; Riboflavin complex; Oxidation-reduction potential; Hydrogen bonding; Nuclear magnetic resonance spectroscopy; Desulfovibrio vulgaris;
Reversible ischemic inhibition of F1F0-ATPase in rat and human myocardium by Kari Ylitalo; Antti Ala-Rämi; Klaus Vuorinen; Keijo Peuhkurinen; Martti Lepojärvi; Päivi Kaukoranta; Kai Kiviluoma; Ilmo Hassinen (329-339).
The physiological role of F1F0-ATPase inhibition in ischemia may be to retard ATP depletion although views of the significance of IF1 are at variance. We corroborate here a method for measuring the ex vivo activity of F1F0-ATPase in perfused rat heart and show that observation of ischemic F1F0-ATPase inhibition in rat heart is critically dependent on the sample preparation and assay conditions, and that the methods can be applied to assay the ischemic and reperfused human heart during coronary by-pass surgery. A 5-min period of ischemia inhibited F1F0-ATPase by 20% in both rat and human myocardium. After a 15-min reperfusion a subsequent 5-min period of ischemia doubled the inhibition in the rat heart but this potentiation was lost after 120 min of reperfusion. Experiments with isolated rat heart mitochondria showed that ATP hydrolysis is required for effective inhibition by uncoupling. The concentration of oligomycin for 50% inhibition (I50) for oxygen consumption was five times higher than its I50 for F1F0-ATPase. Because of the different control strengths of F1F0-ATPase in oxidative phosphorylation and ATP hydrolysis an inhibition of the F1F0-ATPase activity in ischemia with the resultant ATP-sparing has an advantage even in an ischemia/reperfusion situation.
Keywords: Ischemia; Mitochondrial F1F0-ATPase; IF1; Preconditioning;
Identification of Lhcb1/Lhcb2/Lhcb3 heterotrimers of the main light-harvesting chlorophyll a/b–protein complex of Photosystem II (LHC II) by Grzegorz Jackowski; Karol Kacprzak; Stefan Jansson (340-345).
Using non-denaturing isoelectric focusing in polyacrylamide vertical slab gel, we have purified to homogeneity three trimeric subcomplexes of LHC II from Arabidopsis thylakoid membranes. The polypeptide composition of the subcomplexes were studied by immunoblotting. Our results indicate the existence in vivo of LHC II heterotrimers containing Lhcb1, Lhcb2 and Lhcb3 gene products.
Keywords: Light harvesting complex II; Subcomplex; Trimer; (Arabidopsis thaliana);
Heterocyclic ortho-quinones, a novel type of Photosystem II inhibitors by Walter Oettmeier; Klaus Masson; Hans-Jürgen Hecht (346-351).
Members of the new chemical class of 7-substituted 6-bromo-benzo[4,5]imidazo[1,2α]pyridin-8,9-diones were found to be excellent inhibitors at the QB site of the photosystem II D1 reaction center protein. The best inhibitors with pI 50-values of >7 are: dimethyl-propyl, 7.05; i-pentyl, 7.36; t. butyl, 7.47; and i-propyl, 7.51. Displacement experiments with [14C]atrazine revealed that the 8,9-diones behave non-competitively in respect of Photosystem II herbicides and, hence, have to be considered as a new type of Photosystem II inhibitors. This notion is further corroborated by their inhibitory activity in D1 mutants of Chlamydomonas reinhardtii.
Keywords: 7-Substituted 6-bromo-benzo[4,5]imidazo[1,2α]pyridin-8,9-dione; Dichlorophenol–indophenol reduction; [14C]Atrazine displacement; QB binding site;
Transmembrane orientation and topology of the NADH:quinone oxidoreductase putative quinone binding subunit NuoH by Robert Roth; Cecilia Hägerhäll (352-362).
NADH:quinone oxidoreductase, or Complex I, is a multi-subunit membrane-bound enzyme in the respiratory chain of many pro- and eukaryotes. The enzyme catalyzes the oxidation of NADH and donates electrons to the quinone pool, coupled to proton translocation across the membrane, but the mechanism of energy transduction is not understood. In bacteria the enzyme consists of 14 subunits, seven membrane spanning and seven protruding from the membrane. The hydrophobic NuoH (NQO8, ND1, NAD1, NdhA) subunit is seemingly involved in quinone binding. A homologous, structurally and most likely functionally similar subunit is also found in F420H2 oxidoreductases and in complex membrane-bound hydrogenases. We have made theoretical analyses of NuoH and NuoH-like polypeptides and experimentally analyzed the transmembrane topology of the NuoH subunit from Rhodobacter capsulatus by constructing and analyzing alkaline phosphatase fusion proteins. This demonstrated that the NuoH polypeptide has eight transmembrane segments, and four highly conserved hydrophilic sequence motifs facing the inside, bacterial cytoplasm. The N-terminal and C-terminal ends are located on the outside of the membrane. A topology model of NuoH based on these results is presented, and implications from the model are discussed.
Keywords: Complex I; NADH:quinone oxidoreductase; Hydrogenase; F420; NuoH; ND1; NQO8; NAD1; NdhA; Alkaline phosphatase fusion; (Rhodobacter capsulatus);
Efficient large scale purification of his-tagged proton translocating NADH:ubiquinone oxidoreductase (complex I) from the strictly aerobic yeast Yarrowia lipolytica by Noushin Kashani-Poor; Stefan Kerscher; Volker Zickermann; Ulrich Brandt (363-370).
Proton translocating NADH:ubiquinone oxidoreductase (complex I) is the largest membrane bound multiprotein complex of the respiratory chain and the only one for which no molecular structure is available so far. Thus, information on the mechanism of this central enzyme of aerobic energy metabolism is still very limited. As a new approach to analyze complex I, we have recently established the strictly aerobic yeast Yarrowia lipolytica as a model system that offers a complete set of convenient genetic tools and contains a complex I that is stable after isolation. For crystallization of complex I and to obtain its molecular structure it is a prerequisite to prepare large amounts of highly pure enzyme. Here we present the construction of his-tagged complex I that for the first time allows efficient affinity purification. Our protocol recovers almost 40% of complex I present in Yarrowia mitochondrial membranes. Overall, 40–80 mg highly pure and homogeneous complex I can be obtained from 10 l of an overnight Y. lipolytica culture. After reconstitution into asolectin proteoliposomes, the purified enzyme exhibits full NADH:ubiquinone oxidoreductase activity, is fully sensitive to inhibition by quinone analogue inhibitors and capable of generating a proton-motive force.
Keywords: Mitochondria; NADH; Complex I; Yeast; Affinity purification; Proteoliposome; Yarrowia lipolytica;
Origin and properties of fluorescence emission from the extrinsic 33 kDa manganese stabilizing protein of higher plant water oxidizing complex by T. Shutova; G. Deikus; K.-D. Irrgang; V.V. Klimov; G. Renger (371-378).
The fluorescence properties of the isolated extrinsic 33 kDa subunit acting as ‘manganese stabilizing protein’ (MSP) of the water oxidizing complex in photosynthesis was analyzed in buffer solution. Measurements of the emission spectra as a function of excitation wavelength, pH and temperature led to the following results: (a) under all experimental conditions the spectra monitored were found to be the composite of two contributions referred to as ‘306 nm band’ and ‘long-wavelength band’, (b) the excitation spectra of these two bands closely resemble those of tyrosine and tryptophan in solution, respectively, (c) the spectral shape of the ‘306 nm band’ is virtually independent on pH but its amplitude drastically decreases in the alkaline with a pK of 11.7, (d) the amplitude of the ‘long-wavelength’ emission band at alkaline pH slightly increases when the pH rises from 7.2 to about 11.3 followed by a sharp decline at higher pH, and (e) the shape of the overall spectrum at pH 7.2 is only slightly changed upon heating to 90°C whereas the amplitude significantly declines. Based on these findings the two distinct fluorescence bands are ascribed to tyrosine(s) (‘306 nm band’) and the only tryptophan residue W241 of MSP from higher plants (‘long-wavelength band’) as emitters which are both embedded into a rather hydrophobic environment.
Keywords: Photosystem II; Manganese stabilizing protein; Fluorescence; pH dependence;
Functional complexes of mitochondria with Ca,MgATPases of myofibrils and sarcoplasmic reticulum in muscle cells by Enn K Seppet; Tuuli Kaambre; Peeter Sikk; Toomas Tiivel; Heiki Vija; Michael Tonkonogi; Kent Sahlin; Laurence Kay; Florence Appaix; Urmo Braun; Margus Eimre; Valdur A Saks (379-395).
Regulation of mitochondrial respiration in situ in the muscle cells was studied by using fully permeabilized muscle fibers and cardiomyocytes. The results show that the kinetics of regulation of mitochondrial respiration in situ by exogenous ADP are very different from the kinetics of its regulation by endogenous ADP. In cardiac and m. soleus fibers apparent K m for exogenous ADP in regulation of respiration was equal to 300–400 μM. However, when ADP production was initiated by intracellular ATPase reactions, the ADP concentration in the medium leveled off at about 40 μM when about 70% of maximal rate of respiration was achieved. Respiration rate maintained by intracellular ATPases was suppressed about 20–30% during exogenous trapping of ADP with excess pyruvate kinase (PK, 20 IU/ml) and phosphoenolpyruvate (PEP, 5 mM). ADP flux via the external PK+PEP system was decreased by half by activation of mitochondrial oxidative phosphorylation. Creatine (20 mM) further activated the respiration in the presence of PK+PEP. It is concluded that in oxidative muscle cells mitochondria behave as if they were incorporated into functional complexes with adjacent ADP producing systems – with the MgATPases in myofibrils and Ca,MgATPases of sarcoplasmic reticulum.
Keywords: Muscle; Respiration; Mitochondrion; ADP; ATPase;
Changes of absorption spectra during heat-induced denaturation of Photosystem II core antenna complexes CP43 and CP47: revealing the binding states of chlorophyll molecules in these two complexes by Jixiu Shan; Jushuo Wang; Xiang Ruan; Liangbi Li; Yandao Gong; Nanming Zhao; Tingyun Kuang (396-408).
The Photosystem II (PSII) core antenna complexes, CP43 and CP47, were prepared from spinach (Spinacia oleracea L.). The absorption spectra in the red region at room temperature were recorded for the PSII core antenna samples after increased temperature treatment (up to 80°C). Derivative and difference spectra revealed the existence of two groups of chlorophyll a (Chl a) molecules in both CP43 and CP47. The one with the absorption peak in the shorter wavelength region was designated as CP43-669 and CP47-669, while the other with the absorption peak in the longer wavelength region was designated as CP43-682 and CP47-680. The results of the thermal treatment experiment demonstrated that CP43-669 and CP47-669 may exist as monomers of Chl a and that their binding sites on the polypeptides are insensitive to thermal treatment, whereas CP43-682 and CP47-680 may exist as dimers or multimers of Chl a and their binding regions in the polypeptide chains are more sensitive to heat treatment. The excitation energy transfer mechanism between these two different groups of Chl a molecules is also analyzed.
Keywords: Chlorophyll binding protein complex 43; Chlorophyll binding protein complex 47; Chlorophyll a; Heat denaturation; Binding state; Energy transfer;
The manganese stabilizing protein (MSP) and the control of O2 evolution in the unicellular, diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142 by Don L. Tucker; Kim Hirsh; Hong Li; Bethany Boardman; Louis A. Sherman (409-422).
The unicellular diazotrophic cyanobacterium, Cyanothece sp. ATCC 51142 temporally separates N2 fixation from photosynthesis. To better understand the processes by which photosynthesis is regulated, we have analyzed Photosystem (PS) II O2 evolution and the PSII lumenal proteins, especially the Mn stabilizing protein (MSP). We describe a procedure using glycine betaine to isolate photosynthetic membranes from Cyanothece sp. that have high rates of PSI and PSII activity. Analysis with these membranes demonstrated similar patterns of O2 evolution in vivo and in vitro, with a trough at the time of maximal N2 fixation and with a peak in the late light period. The pattern of PSI activity was also similar in vivo and in vitro. We cloned the genes for MSP (psbO) and the 12 kDa protein (psbU) and analyzed their transcriptional properties throughout the diurnal cycle. We suggest that the changes in PSII activity in Cyanothece sp. were due to conformational changes in a highly flexible MSP, a suggestion which can now be studied in a chimera. The Cyanothece sp. psbO gene has been transformed into Synechocystis sp. PCC 6803; MSP and O2 evolution in the resulting transformant had properties that were similar to those in Cyanothece sp., providing additional confirmation for the properties of Cyanothece sp. MSP.
Keywords: Cyanobacteria; Disulfide bonds; Nitrogen fixation; Oxygen evolution; Mn-stabilizing protein; Photosynthesis;
Plasma membrane electron transport coupled to Na+ extrusion in the halotolerant alga Dunaliella by Adriana Katz; Uri Pick (423-431).
The halotolerant alga Dunaliella adapts to exceptionally high salinity and maintains low [Na+]in at hypersaline solutions, suggesting that it possesses efficient mechanisms for regulating intracellular Na+. In this work we examined the possibility that Na+ export in Dunaliella is linked to a plasma membrane electron transport (redox) system. Na+ extrusion was induced in Dunaliella cells by elevation of intracellular Na+ with Na+-specific ionophores. Elevation of intracellular Na+ was found to enhance the reduction of an extracellular electron acceptor ferricyanide (FeCN). The quinone analogs NQNO and dicumarol inhibited FeCN reduction and led to accumulation of Na+ by inhibition of Na+ extrusion. These inhibitors also diminished the plasma membrane potential in Dunaliella. Anaerobic conditions elevated, whereas FeCN partially decreased intracellular Na+ content. Cellular NAD(P)H level decreased upon enhancement of plasma membrane electron transport. These results are consistent with the operation of an electrogenic NAD(P)H-driven redox system coupled to Na+ extrusion in Dunaliella plasma membrane. We propose that redox-driven Na+ extrusion and recycling in Dunaliella evolved as means of adaptation to hypersaline environments.
Keywords: Na+ extrusion; Na+ homeostasis; Plasma membrane electron transport; Salt tolerance; (Dunaliella);
Accessibility of tyrosine Y⋅ Z to exogenous reductants and Mn2+ in various Photosystem II preparations by Stamatia Chroni; Demetrios F. Ghanotakis (432-437).
The reduction of tyrosine Y⋅ Z by benzidine and exogenous Mn2+ was studied by kinetic EPR experiments in various Photosystem II (PSII) preparations. Using lanthanide treated PSII membranes it was demonstrated that neither the extrinsic polypeptides (17, 23 and 33 kDa) nor the Mn complex block the accessibility of Y⋅ Z to exogenous reductants, such as benzidine. In addition, it was shown that in the presence of the native Mn complex exogenous Mn2+ does not reduce Y⋅ Z.
Keywords: Photosystem II; Tyrosine YZ; Exogenous reductant; Manganese complex;
Adenylate kinase activity in rod outer segments of bovine retina by Luigi Notari; Isidoro Mario Pepe; Carlo Cugnoli; Alessandro Morelli (438-443).
The rod outer segments of bovine retina contain two different adenylate kinases: a soluble activity, which is not sensitive to calcium ion, and an activity bound to disk membranes, which is dependent on the calcium levels. In fact, the maximal activity associated to the disks is reached at Ca2+ concentrations between 10−6 and 10−7 M, which is the range of calcium level actually present in the rod cell. The Michaelis-Menten kinetics of the enzyme activity on disk membranes was determined and the actual concentrations of ATP, AMP and ADP were measured in the photoreceptor outer segment. Therefore, the physiological relevance of the adenylate kinase activity was discussed considering the above results. The formation of ATP catalyzed by the enzyme seems appropriate to supply at least some of the reactions necessary for phototransduction, indicating that ATP could be regenerated from ADP directly on the disk membranes where the photoreception events take place.
Keywords: Retina; Phototransduction; Rod outer segment; Adenylate kinase; Vision;
The energy level scheme for the ferryl heme in compound II of the peroxidase-catalase family as determined from analysis of low-temperature magnetic circular dichroism by Yuri A. Sharonov (444-451).
The expressions for temperature-dependent magnetic circular dichroism (MCD) of the ferryl heme (Fe4+Por, S=1), which is a model of an intermediate product of the catalytic cycle of heme enzymes (compound II), have been derived in the framework of a two-term model. Theoretical predictions for the temperature and magnetic field dependence of MCD intensity of the ferryl heme are compared with those of the high-spin and low-spin ferric heme. Analysis of reported MCD spectra of myoglobin peroxide [Foot et al., Biochem. J. 2651 (1989) 515–522] and compound II of horseradish peroxidase [Browett et al., J. Am. Chem. Soc. 110 (1987) 3633–3640] has shown the presence in the samples of ∼1% of a low-spin ferric component, which, however, should be taken into account in simulating observed temperature dependences of MCD intensity. The values of two adjustable parameters are estimated from the fit of the observed and simulated plots of MCD intensity against the reciprocal of the absolute temperature. One of them, the energy gap between the ground and excited terms, predetermines the axial zero-field splitting. The other parameter is correlated with the energy of splitting of excited quartets arising from either the porphyrin π→π* transition or the spin-allowed charge-transfer transition.
Keywords: Magnetic circular dichroism; Ferryl heme; Compound II; Hemoprotein; Theoretical model;
Author Index (452-454).
Information for Contributors (457-462).