BBA - Bioenergetics (v.1787, #4)
Editorial Board (i).
P-type ATPases as drug targets: Tools for medicine and science by Laure Yatime; Morten J. Buch-Pedersen; Maria Musgaard; J. Preben Morth; Anne-Marie Lund Winther; Bjørn P. Pedersen; Claus Olesen; Jens Peter Andersen; Bente Vilsen; Birgit Schiøtt; Michael G. Palmgren; Jesper V. Møller; Poul Nissen; Natalya Fedosova (207-220).
P-type ATPases catalyze the selective active transport of ions like H+, Na+, K+, Ca2+, Zn2+, and Cu2+ across diverse biological membrane systems. Many members of the P-type ATPase protein family, such as the Na+,K+-, H+,K+-, Ca2+-, and H+-ATPases, are involved in the development of pathophysiological conditions or provide critical function to pathogens. Therefore, they seem to be promising targets for future drugs and novel antifungal agents and herbicides. Here, we review the current knowledge about P-type ATPase inhibitors and their present use as tools in science, medicine, and biotechnology. Recent structural information on a variety of P-type ATPase family members signifies that all P-type ATPases can be expected to share a similar basic structure and a similar basic machinery of ion transport. The ion transport pathway crossing the membrane lipid bilayer is constructed of two access channels leading from either side of the membrane to the ion binding sites at a central cavity. The selective opening and closure of the access channels allows vectorial access/release of ions from the binding sites. Recent structural information along with new homology modeling of diverse P-type ATPases in complex with known ligands demonstrate that the most proficient way for the development of efficient and selective drugs is to target their ion transport pathway.
Keywords: P-type ATPases; Pump; Inhibitor; Ion transport pathway; Structure-based drug design;
The chemistry of the CuB site in cytochrome c oxidase and the importance of its unique His–Tyr bond by Ville R.I. Kaila; Mikael P. Johansson; Dage Sundholm; Liisa Laakkonen; Mårten Wikström (221-233).
The CuB metal center is at the core of the active site of the heme–copper oxidases, comprising a copper atom ligating three histidine residues one of which is covalently bonded to a tyrosine residue. Using quantum chemical methodology, we have studied the CuB site in several redox and ligand states proposed to be intermediates of the catalytic cycle. The importance of the His–Tyr crosslink was investigated by comparing energetics, charge, and spin distributions between systems with and without the crosslink. The His–Tyr bond was shown to decrease the proton affinity and increase the electron affinity of both Tyr-244 and the copper. A previously unnoticed internal electronic equilibrium between the copper atom and the tyrosine was observed, which seems to be coupled to the unique structure of the system. In certain states the copper and Tyr-244 compete for the unpaired electron, the localization of which is determined by the oxygenous ligand of the copper. This electronic equilibrium was found to be sensitive to the presence of a positive charge 10 Å away from the center, simulating the effect of Lys-319 in the K-pathway of proton transfer. The combined results provide an explanation for why the heme–copper oxidases need two pathways of proton uptake, and why the K-pathway is active only in the second half of the reaction cycle.
Keywords: Oxygen reduction; Proton transfer; DFT; Quantum chemistry;
Characterisation of the electron transfer and complex formation between Flavodoxin from D. vulgaris and the haem domain of Cytochrome P450 BM3 from B. megaterium by Andrea Fantuzzi; Yergalem T. Meharenna; Paul B. Briscoe; Francoise Guerlesquin; Sheila J. Sadeghi; Gianfranco Gilardi (234-241).
Investigation of the complex formation and electron transfer kinetics between P450 BMP and flavodoxin was carried out following the suggested involvement of flavodoxin in modulating the electron transfer to BMP in artificial redox chains bound to an electrode surface. While electron transfer measurements show the formation of a tightly bound complex, the NMR data indicate the formation of shortly lived complexes. The measured k obs ranged from 24.2 s− 1 to 44.1 s− 1 with k on ranging from 0.07 × 106 to 1.1 × 106 s− 1M− 1 and K d ranging from 300 μM to 24 μM in buffers of different ionic strength. This apparent contradiction is due to the existence of two events in the complex formation prior to electron transfer. A stable complex is initially formed. Within such tightly bound complex, flavodoxin rocks rapidly between different positions. The rocking of the bound flavodoxin between several different orientations gives rise to the transient complexes in fast exchange as observed in the NMR experiments. Docking simulations with two different approaches support the theory that there is no highly specific orientation in the complex, but instead one side of the flavodoxin binds the P450 with high overall affinity but with a number of different orientations. The level of functionality of each orientation is dependent on the distance between cofactors, which can vary between 8 and 25 Å, with some of the transient complexes showing distances compatible with the measured electron transfer rate constants.
Keywords: Electron transfer; Flavodoxin; P450 BMP; Transient complex; Protein–protein docking;
Assembly of subunit d (Vma6p) and G (Vma10p) and the NMR solution structure of subunit G (G1–59) of the Saccharomyces cerevisiae V1VO ATPase by Sankaranarayanan Rishikesan; Shovanlal Gayen; Youg R. Thaker; Subramanian Vivekanandan; Malathy S.S. Manimekalai; Yin Hoe Yau; Susana Geifman Shochat; Gerhard Grüber (242-251).
Understanding the structural traits of subunit G is essential, as it is needed for V1VO assembly and function. Here solution NMR of the recombinant N- (G1–59) and C-terminal segment (G61–114) of subunit G, has been performed in the absence and presence of subunit d of the yeast V-ATPase. The data show that G does bind to subunit d via its N-terminal part, G1–59 only. The residues of G1–59 involved in d binding are Gly7 to Lys34. The structure of G1–59 has been solved, revealing an α-helix between residues 10 and 56, whereby the first nine- and the last three residues of G1–59 are flexible. The surface charge distribution of G1–59 reveals an amphiphilic character at the N-terminus due to positive and negative charge distribution at one side and a hydrophobic surface on the opposite side of the structure. The C-terminus exhibits a strip of negative residues. The data imply that G1–59–d assembly is accomplished by hydrophobic interactions and salt-bridges of the polar residues. Based on the recently determined NMR structure of segment E18–38 of subunit E of yeast V-ATPase and the presently solved structure of G1–59, both proteins have been docked and binding epitopes have been analyzed.
Keywords: Vacuolar-type ATPase; V1VO ATPase; Subunit d; Subunit G; Subunit E; Vma6p; Vma7p; Vma10p;
Electron transfer protein complexes in the thylakoid membranes of heterocysts from the cyanobacterium Nostoc punctiforme by Tanai Cardona; Natalia Battchikova; Pengpeng Zhang; Karin Stensjö; Eva-Mari Aro; Peter Lindblad; Ann Magnuson (252-263).
Filamentous, heterocystous cyanobacteria are capable of nitrogen fixation and photoautotrophic growth. Nitrogen fixation takes place in heterocysts that differentiate as a result of nitrogen starvation. Heterocysts uphold a microoxic environment to avoid inactivation of nitrogenase, e.g. by downregulation of oxygenic photosynthesis. The ATP and reductant requirement for the nitrogenase reaction is considered to depend on Photosystem I, but little is known about the organization of energy converting membrane proteins in heterocysts. We have investigated the membrane proteome of heterocysts from nitrogen fixing filaments of Nostoc punctiforme sp. PCC 73102, by 2D gel electrophoresis and mass spectrometry. The membrane proteome was found to be dominated by the Photosystem I and ATP-synthase complexes. We could identify a significant amount of assembled Photosystem II complexes containing the D1, D2, CP43, CP47 and PsbO proteins from these complexes. We could also measure light-driven in vitro electron transfer from Photosystem II in heterocyst thylakoid membranes. We did not find any partially disassembled Photosystem II complexes lacking the CP43 protein. Several subunits of the NDH-1 complex were also identified. The relative amount of NDH-1M complexes was found to be higher than NDH-1L complexes, which might suggest a role for this complex in cyclic electron transfer in the heterocysts of Nostoc punctiforme.
Keywords: Cyanobacteria; Nostoc; Heterocyst; Photosystem II; Fluorescence; BN-PAGE;
Regulation of Acanthamoeba castellanii alternative oxidase activity by mutual exclusion of purine nucleotides; ATP's inhibitory effect by Andrzej M. Woyda-Ploszczyca; Francis E. Sluse; Wieslawa Jarmuszkiewicz (264-271).
The effects of different adenine and guanine nucleotides on the cyanide-resistant respiration (i.e. alternative oxidase (AcAOX) activity) of mitochondria from the amoeba A. castellanii mitochondria were studied. We found that guanine nucleotides activate AcAOX to a greater degree than adenine nucleotides, and that nucleoside monophosphates were more efficient activators than nucleoside di- or triphosphates. The extent of the nucleotides' influence on AcAOX was dependent on the medium's pH and was more pronounced at pH 6.8, which is optimal for AcAOX activity. In contrast to other purine nucleosides, we demonstrate, for the first time, that ATP has an inhibitory effect on AcAOX activity. Since we also observed the inhibition by ATP in the mitochondria of another protozoon, such as Dictyostelium discoideum, and the yeast, Candida maltosa, it may be a regulatory feature common to all purine nucleotide-modulated non-plant AOXs. The physiological importance of this discovery is discussed. Kinetic data show that the binding of GMP (a positive allosteric effector) and the binding of ATP (a negative allosteric effector) to AcAOX are mutually exclusive. ATP's inhibition of the enzyme can be overcome by sufficiently high concentrations of GMP, and conversely, GMP's stimulation can be overcome by sufficiently high concentrations of ATP. However, an approximately three times lower concentration of GMP compared to ATP gives a half maximal effect on AcAOX activity. This is indicative of a higher binding affinity for the positive effector at the same or, at least overlapping, nucleotide-binding sites on AcAOX. These results suggest that AcAOX activity in A. castellanii mitochondria might be controlled by the relative intracellular concentrations of purine nucleotides.
Keywords: Alternative oxidase; Mitochondria; Purine nucleotides; Acanthamoeba castellanii; Kinetics; Mutual exclusion;
Structural organisation of phycobilisomes from Synechocystis sp. strain PCC6803 and their interaction with the membrane by Ana A. Arteni; Ghada Ajlani; Egbert J. Boekema (272-279).
In cyanobacteria, the harvesting of light energy for photosynthesis is mainly carried out by the phycobilisome — a giant, multi-subunit pigment–protein complex. This complex is composed of heterodimeric phycobiliproteins that are assembled with the aid of linker polypeptides such that light absorption and energy transfer to photosystem II are optimised. In this work we have studied, using single particle electron microscopy, the phycobilisome structure in mutants lacking either two or all three of the phycocyanin hexamers. The images presented give much greater detail than those previously published, and in the best two-dimensional projection maps a resolution of 13 Å was achieved. As well as giving a better overall picture of the assembly of phycobilisomes, these results reveal new details of the association of allophycocyanin trimers within the core. Insights are gained into the attachment of this core to the membrane surface, essential for efficient energy transfer to photosystem II. Comparison of projection maps of phycobilisomes with and without reconstituted ferredoxin:NADP oxidoreductase suggests a location for this enzyme within the complex at the rod-core interface.
Keywords: Cyanobacteria; Phycobilisome; Ferredoxin; NADP oxidoreductase; Electron microscopy; Negative staining; Single particle image analysis;
Influence of zeaxanthin and echinenone binding on the activity of the Orange Carotenoid Protein by Claire Punginelli; Adjélé Wilson; Jean-Marc Routaboul; Diana Kirilovsky (280-288).
In most cyanobacteria high irradiance induces a photoprotective mechanism that downregulates photosynthesis by increasing thermal dissipation of the energy absorbed by the phycobilisome, the water-soluble antenna. The light activation of a soluble carotenoid protein, the Orange-Carotenoid-Protein (OCP), binding hydroxyechinenone, a keto carotenoid, is the key inducer of this mechanism. Light causes structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. Here, we tested whether echinenone or zeaxanthin can replace hydroxyechinenone in a study in which the nature of the carotenoid bound to the OCP was genetically changed. In a mutant lacking hydroxyechinenone and echinenone, the OCP was found to bind zeaxanthin but the stability of the binding appeared to be lower and light was unable to photoconvert the dark form into a red active form. Moreover, in the strains containing zeaxanthin-OCP, blue-green light did not induce the photoprotective mechanism. In contrast, in mutants in which echinenone is bound to the OCP, the protein is photoactivated and photoprotection is induced. Our results strongly suggest that the presence of the carotenoid carbonyl group that distinguishes echinenone and hydroxyechinenone from zeaxanthin is essential for the OCP activity.
Keywords: Cyanobacteria; Non-photochemical quenching; Orange-Carotenoid-Protein; Photoprotection; Photosystem II; Synechocystis;