BBA - Bioenergetics (v.1767, #5)
Editorial Board (ii).
Structure, function and regulation of plant photosystem I by Poul Erik Jensen; Roberto Bassi; Egbert J. Boekema; Jan P. Dekker; Stefan Jansson; Dario Leister; Colin Robinson; Henrik Vibe Scheller (335-352).
Photosystem I (PSI) is a multisubunit protein complex located in the thylakoid membranes of green plants and algae, where it initiates one of the first steps of solar energy conversion by light-driven electron transport. In this review, we discuss recent progress on several topics related to the functioning of the PSI complex, like the protein composition of the complex in the plant Arabidopsis thaliana, the function of these subunits and the mechanism by which nuclear-encoded subunits can be inserted into or transported through the thylakoid membrane. Furthermore, the structure of the native PSI complex in several oxygenic photosynthetic organisms and the role of the chlorophylls and carotenoids in the antenna complexes in light harvesting and photoprotection are reviewed. The special role of the ‘red’ chlorophylls (chlorophyll molecules that absorb at longer wavelength than the primary electron donor P700) is assessed. The physiology and mechanism of the association of the major light-harvesting complex of photosystem II (LHCII) with PSI during short term adaptation to changes in light quality and quantity is discussed in functional and structural terms. The mechanism of excitation energy transfer between the chlorophylls and the mechanism of primary charge separation is outlined and discussed. Finally, a number of regulatory processes like acclimatory responses and retrograde signalling is reviewed with respect to function of the thylakoid membrane. We finish this review by shortly discussing the perspectives for future research on PSI.
Keywords: Excitation energy transfer; Light harvesting; Photosynthesis; Red chlorophylls; Regulation; State transitions;
Macromolecular crowding and its influence on possible reaction mechanisms in photosynthetic electron flow by I.G. Tremmel; E. Weis; G.D. Farquhar (353-361).
The diffusion of plastoquinol and its binding to the Qo site of the cyt bf complex in the course of photosynthetic electron transport was studied by following the sigmoidal flash-induced re-reduction kinetics of P700 after previous oxidation of the intersystem electron carriers. The data resulting from these experiments were matched with a simulation of electron transport using Monte Carlo techniques. The simulation was able to account for the experimental observations. Two different extreme cases of reaction mechanism at the Qo site were compared: a diffusion limited collisional mechanism and a non-diffusion limited tight binding mechanism. Assuming a tight binding mechanism led to best matches due to the high protein density in thylakoids. The varied parameters resulted in values well within the range of published data. The results emphasise the importance of structural characteristics of thylakoids in models of electron transport.
Keywords: Photosynthesis; Thylakoid membranes; Macromolecular crowding; Reaction mechanism; Plastoquinone diffusion; Monte Carlo simulation;
A pathway for protons in nitric oxide reductase from Paracoccus denitrificans by Joachim Reimann; Ulrika Flock; Håkan Lepp; Alf Honigmann; Pia Ädelroth (362-373).
Nitric oxide reductase (NOR) from P. denitrificans is a membrane-bound protein complex that catalyses the reduction of NO to N2O (2NO + 2e− + 2H+ → N2O + H2O) as part of the denitrification process. Even though NO reduction is a highly exergonic reaction, and NOR belongs to the superfamily of O2-reducing, proton-pumping heme-copper oxidases (HCuOs), previous measurements have indicated that the reaction catalyzed by NOR is non-electrogenic, i.e. not contributing to the proton electrochemical gradient. Since electrons are provided by donors in the periplasm, this non-electrogenicity implies that the substrate protons are also taken up from the periplasm. Here, using direct measurements in liposome-reconstituted NOR during reduction of both NO and the alternative substrate O2, we demonstrate that protons are indeed consumed from the ‘outside’. First, multiple turnover reduction of O2 resulted in an increase in pH on the outside of the NOR-vesicles. Second, comparison of electrical potential generation in NOR-liposomes during oxidation of the reduced enzyme by either NO or O2 shows that the proton transfer signals are very similar for the two substrates proving the usefulness of O2 as a model substrate for these studies. Last, optical measurements during single-turnover oxidation by O2 show electron transfer coupled to proton uptake from outside the NOR-liposomes with a τ = 15 ms, similar to results obtained for net proton uptake in solubilised NOR [U. Flock, N.J. Watmough, P. Ädelroth, Electron/proton coupling in bacterial nitric oxide reductase during reduction of oxygen, Biochemistry 44 (2005) 10711–10719]. NOR must thus contain a proton transfer pathway leading from the periplasmic surface into the active site. Using homology modeling with the structures of HCuOs as templates, we constructed a 3D model of the NorB catalytic subunit from P. denitrificans in order to search for such a pathway. A plausible pathway, consisting of conserved protonatable residues, is suggested.
Keywords: Proton transfer; Electron transfer; Proteoliposomes; Flow-flash; Non-heme iron; Nitric oxide; Oxygen; Homology modeling; Sequence alignments;
Expression by Chlamydomonas reinhardtii of a chloroplast ATP synthase with polyhistidine-tagged beta subunits by Eric A. Johnson; Julian Rosenberg; Richard E. McCarty (374-380).
The green alga Chlamydomonas reinhardtii is a model organism for the study of photosynthesis. The chloroplast ATP synthase is responsible for the synthesis of ATP during photosynthesis. Using genetic engineering and biolistic transformation, a string of eight histidine residues has been inserted into the amino-terminal end of the β subunit of this enzyme in C. reinhardtii. The incorporation of these amino acids did not impact the function of the ATP synthase either in vivo or in vitro and the resulting strain of C. reinhardtii showed normal growth. The addition of these amino acids can be seen through altered gel mobility of the β subunit and the binding of a polyhistidine-specific dye to the subunit. The purified his-tagged CF1 has normal Mg2+-ATPase activity, which can be stimulated by alcohol and detergents and the enzyme remains active while bound to a nickel-coated surface. Potential uses for this tagged enzyme as a biochemical tool are discussed.
Keywords: Chlamydomonas reinhardtii; ATP synthase; CF1; Polyhistidine; His-tag;
The inside pH determines rates of electron and proton transfer in vesicle-reconstituted cytochrome c oxidase by Kristina Faxén; Peter Brzezinski (381-386).
Cytochrome c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria where it translocates protons across a membrane thereby maintaining an electrochemical proton gradient. Results from earlier studies on detergent-solubilized cytochrome c oxidase have shown that individual reaction steps associated with proton pumping display pH-dependent kinetics. Here, we investigated the effect of pH on the kinetics of these reaction steps with membrane-reconstituted cytochrome c oxidase such that the pH was adjusted to different values on the inside and outside of the membrane. The results show that the pH on the inside of the membrane fully determines the kinetics of internal electron transfers that are linked to proton pumping. Thus, even though proton release is rate limiting for these reaction steps (Salomonsson et al., Proc. Natl. Acad. Sci. USA, 2005, 102, 17624), the transition kinetics is insensitive to the outside pH (in the range 6–9.5).
Keywords: Rhodobacter sphaeroides; Proton pumping; Cytochrome aa 3; Respiration; Kinetics;
Accommodation of NO in the active site of mammalian and bacterial cytochrome c oxidase aa 3 by Eric Pilet; Wolfgang Nitschke; Ursula Liebl; Marten H. Vos (387-392).
Following different reports on the stoichiometry and configuration of NO binding to mammalian and bacterial reduced cytochrome c oxidase aa 3 (CcO), we investigated NO binding and dynamics in the active site of beef heart CcO as a function of NO concentration, using ultrafast transient absorption and EPR spectroscopy. We find that in the physiological range only one NO molecule binds to heme a 3, and time-resolved experiments indicate that even transient binding to CuB does not occur. Only at very high (∼ 2 mM) concentrations a second NO is accommodated in the active site, although in a different configuration than previously observed for CcO from Paracoccus denitrificans [E. Pilet, W. Nitschke, F. Rappaport, T. Soulimane, J.-C. Lambry, U. Liebl and M.H. Vos. Biochemistry 43 (2004) 14118–14127], where we proposed that a second NO does bind to CuB. In addition, in the bacterial enzyme two NO molecules can bind already at NO concentrations of ∼ 1 μM. The unexpected differences highlighted in this study may relate to differences in the physiological relevance of the CcO–NO interactions in both species.
Keywords: Cytochrome c oxidase; Nitric oxide; Femtosecond spectroscopy; Electron paramagnetic resonance;
Characterization of a subcomplex of mitochondrial NADH:ubiquinone oxidoreductase (complex I) lacking the flavoprotein part of the N-module by Volker Zickermann; Klaus Zwicker; Maja A. Tocilescu; Stefan Kerscher; Ulrich Brandt (393-400).
Mitochondrial NADH:ubiquinone oxidoreductase is the largest and most complicated proton pump of the respiratory chain. Here we report the preparation and characterization of a subcomplex of complex I selectively lacking the flavoprotein part of the N-module. Removing the 51-kDa and the 24-kDa subunit resulted in loss of catalytic activity. The redox centers of the subcomplex could be reduced neither by NADH nor NADPH demonstrating that physiological electron input into complex I occurred exclusively via the N-module and that the NADPH binding site in the 39-kDa subunit and further potential nucleotide binding sites are isolated from the electron transfer pathway within the enzyme. Taking advantage of the selective removal of two of the eight iron–sulfur clusters of complex I and providing additional evidence by redox titration and site-directed mutagenesis, we could for the first time unambiguously assign cluster N1 of fungal complex I to mammalian cluster N1b.
Keywords: Complex I; NADH:ubiquinone oxidoreductase; Subcomplex; Yarrowia lipolytica; Iron–sulfur cluster; N-module; NADH binding;