BBA - Bioenergetics (v.1707, #2-3)
Editorial Board (ii).
MT FdR: a ferredoxin reductase from M. tuberculosis that couples to MT CYP51 by Allison Zanno; Nicholas Kwiatkowski; Alfin D.N. Vaz; Hebe M. Guardiola-Diaz (157-169).
We report the molecular cloning, expression and partial characterization of MT FdR, an FAD-associated flavoprotein, from Mycobacterium tuberculosis similar to the oxygenase-coupled NADH-dependent ferredoxin reductases (ONFR). We establish, through kinetic and spectral analysis, that MT FdR preferentially uses NADH as cofactor. Furthermore, MT FdR forms a complex with mycobacterial ferredoxin (MT Fdx) and MT CYP51, a cytochrome P450 (CYP) from M. tuberculosis that is similar to lanosterol 14α-demethylase isozymes. This reconstituted system transfers electrons from the cofactor to the heme iron of MT CYP51 and effects the demethylation of lanosterol.
Keywords: CYP51; MT Fdx; FdR; MT FdR; FAD; ONFR;
Evidence for dissociation of chlorophyll b from the main light-harvesting complex in the oligomerization state isolated from marine alga, Bryopsis corticulans by Hui Chen; Shihua Shen; Yu Liang; Jing Leng; Mingjuan Tang; Yandao Gong (170-178).
We investigated the composition and organization of chlorophylls in monomers, trimers and oligomers (small aggregates) of the main light-harvesting complex (LHC II) isolated from marine alga, Bryopsis corticulans, using a combination of measurements with reversed-phase high performance liquid chromatography (RP-HPLC) and steady-state spectroscopy of absorption, circular dichroism (CD) and low temperature fluorescence. The composition and organization of the chlorophylls in monomeric and trimeric LHC II were essentially identical to those of LHC II from higher plants. For LHC II oligomers, a large decrease of chlorophyll (Chl) b absorption and of CD signals corresponding to Chl b was consistent with the quantitative analysis of Chl b by RP-HPLC, indicating that oligomerization of the LHC II proteins significantly influenced spectroscopic properties and led to the dissociation of Chl b molecules from LHC II. Our data strongly suggested that protein oligomerization constitutes a structural basis for the decrease of Chl b molecules in LHC II of B. corticulans. The LHC II of B. corticulans might play a photoprotective role with the reduction of the ability of light absorption via alteration of its own structural conformation.
Keywords: Light-harvesting complex; Chlorophyll; Oligomerization; Spectroscopy; Bryopsis corticulan;
The transient complex of poplar plastocyanin with cytochrome f: effects of ionic strength and pH by Christian Lange; Tobias Cornvik; Irene Díaz-Moreno; Marcellus Ubbink (179-188).
The orientation of poplar plastocyanin in the complex with turnip cytochrome f has been determined by rigid-body calculations using restraints from paramagnetic NMR measurements. The results show that poplar plastocyanin interacts with cytochrome f with the hydrophobic patch of plastocyanin close to the heme region on cytochrome f and via electrostatic interactions between the charged patches on both proteins. Plastocyanin is tilted relative to the orientation reported for spinach plastocyanin, resulting in a longer distance between iron and copper (13.9 Å). With increasing ionic strength, from 0.01 to 0.11 M, all observed chemical-shift changes decrease uniformly, supporting the idea that electrostatic forces contribute to complex formation. There is no indication for a rearrangement of the transient complex in this ionic strength range, contrary to what had been proposed earlier on the basis of kinetic data. By decreasing the pH from pH 7.7 to pH 5.5, the complex is destabilized. This may be attributed to the protonation of the conserved acidic patches or the copper ligand His87 in poplar plastocyanin, which are shown to have similar pK a values. The results are interpreted in a two-step model for complex formation.
Keywords: Electron transfer; Photosynthesis; Protein complex; NMR spectroscopy; Transient complex;
Investigation of B-branch electron transfer by femtosecond time resolved spectroscopy in a Rhodobacter sphaeroides reaction centre that lacks the QA ubiquinone by Dmitrij Frolov; Marion C. Wakeham; Elena G. Andrizhiyevskaya; Michael R. Jones; Rienk van Grondelle (189-198).
The dynamics of electron transfer in a membrane-bound Rhodobacter sphaeroides reaction centre containing a combination of four mutations were investigated by transient absorption spectroscopy. The reaction centre, named WAAH, has a mutation that causes the reaction centre to assemble without a QA ubiquinone (Ala M260 to Trp), a mutation that causes the replacement of the HA bacteriopheophytin with a bacteriochlorophyll (Leu M214 to His) and two mutations that remove acidic groups close to the QB ubiquinone (Glu L212 to Ala and Asp L213 to Ala). Previous work has shown that the QB ubiquinone is reduced by electron transfer along the so-called inactive cofactor branch (B-branch) in the WAAH reaction centre (M.C. Wakeham, M.G. Goodwin, C. McKibbin, M.R. Jones, Photo-accumulation of the P+QB − radical pair state in purple bacterial reaction centres that lack the QA ubiquinone, FEBS Letters 540 (2003) 234–240). In the present study the dynamics of electron transfer in the membrane-bound WAAH reaction centre were studied by femtosecond transient absorption spectroscopy, and the data analysed using a compartmental model. The analysis indicates that the yield of QB reduction via the B-branch is approximately 8% in the WAAH reaction centre, consistent with results from millisecond time-scale kinetic spectroscopy. Possible contributions to this yield of the constituent mutations in the WAAH reaction centre and the membrane environment of the complex are discussed.
Keywords: Reaction centre; Electron transfer; B-branch; Mutagenesis; Compartmental analysis; Femtosecond spectroscopy;
The nuclear genes encoding the internal (KlNDI1) and external (KlNDE1) alternative NAD(P)H:ubiquinone oxidoreductases of mitochondria from Kluyveromyces lactis by N. Tarrío; S. Díaz Prado; M.E. Cerdán; M.I. González Siso (199-210).
Cloning, sequence and functional analyses of the Kluyveromyces lactis genes KlNDI1 and KlNDE1 are reported. These genes encode for proteins with high homology to the mitochondrial internal (Ndi1p) and external (Nde1p) alternative NADH:ubiquinone oxidoreductases from Saccharomyces cerevisiae and complement the respective mutations. Analysis of KlNDI1 transcriptional regulation showed that expression of this gene is lower in 2% glucose than in 0.5% glucose or non-fermentable carbon sources. Beta-galactosidase activity values, shown by lacZ fusions of KlNDI1 promoter deletions, suggested that two Adr1p binding sites mediate this carbon source regulation of KlNDI1. The expression of the KlNDE1 gene in S. cerevisiae mutant strains and measurement of respiration with isolated mitochondria showed that the protein encoded by KlNDE1 oxidizes NADPH, this being an important difference with respect to the conventional yeast S. cerevisiae. Moreover, Northern blot experiments using a phosphoglucose isomerase mutant showed that KlNDE1 gene transcription increases with glucose metabolism through the pentose phosphate pathway.
Keywords: NAD(P)H:ubiquinone oxidoreductase; Alternative dehydrogenase; Kluyveromyces lactis; NDI1; NDE1; Mitochondrial respiration;
Composition of complex I from Neurospora crassa and disruption of two “accessory” subunits by Isabel Marques; Margarida Duarte; Joana Assunção; Alexandra V. Ushakova; Arnaldo Videira (211-220).
Respiratory chain complex I of the fungus Neurospora crassa contains at least 39 polypeptide subunits, of which 35 are conserved in mammals. The 11.5 kDa and 14 kDa proteins, homologues of bovine IP15 and B16.6, respectively, are conserved among eukaryotes and belong to the membrane domain of the fungal enzyme. The corresponding genes were separately inactivated by repeat-induced point-mutations, and null-mutant strains of the fungus were isolated. The lack of either subunit leads to the accumulation of distinct intermediates of the membrane arm of complex I. In addition, the peripheral arm of the enzyme seems to be formed in mutant nuo14 but, interestingly, not in mutant nuo11.5. These results and the analysis of enzymatic activities of mutant mitochondria indicate that both polypeptides are required for complex I assembly and function.
Keywords: Mitochondria; Respiratory chain; Complex I; NADH dehydrogenase; Mutant; Neurospora crassa;
Identification and characterisation of a new class of highly specific and potent inhibitors of the mitochondrial pyruvate carrier by John C.W. Hildyard; Carina Ämmälä; Iain D. Dukes; Stephen A. Thomson; Andrew P. Halestrap (221-230).
Two novel thiazolidine compounds, GW604714X and GW450863X, were found to be potent inhibitors of mitochondrial respiration supported by pyruvate but not other substrates. Direct measurement of pyruvate transport into rat liver and yeast mitochondria confirmed that these agents inhibited the mitochondrial pyruvate carrier (MPC) with K i values <0.1 μM. Inhibitor titrations of pyruvate-dependent respiration by heart mitochondria gave values (±S.E.) for the concentration of inhibitor binding sites (pmol per mg protein) and their K i (nM) of 56.0±0.9 and 0.057±0.010 nM for the more hydrophobic GW604714X; for GW450863X the values were 59.9±4.6 and 0.60±0.12 nM. [3H]-methoxy-GW450863X binding was also used to determine the MPC content of the heart, kidney, liver and brain mitochondria giving values of 56, 40, 26 and 20 pmol per mg protein respectively. Binding to yeast mitochondria was <10% of that in rat liver mitochondria, consistent with the slow rate of pyruvate transport into yeast mitochondria. [3H]-methoxy-GW450863X binding was inhibited by GW604714X and by the established MPC inhibitor, UK5099. The absorbance spectra of GW450863X and GW604714X were markedly changed by the addition of β-mercaptoethanol suggesting that the novel inhibitors, like α-cyanocinnamate, possess an activated double bond that attacks a critical cysteine residue on the MPC. However, no labelled protein was detected following SDS-PAGE suggesting that the covalent modification is reversible. GW604714X and GW450863X inhibited l-lactate transport by the plasma membrane monocarboxylate transporter MCT1, but at concentrations more than four orders of magnitude greater than the MPC.
Keywords: Mitochondrial carrier family; Pyruvate metabolism; Transport;
The bioenergetic role of dioxygen and the terminal oxidase(s) in cyanobacteria by Martina Paumann; Günther Regelsberger; Christian Obinger; Günter A. Peschek (231-253).
Keywords: Respiration; Photosynthesis; Oxygen; Terminal oxidase; Proton-pumping; Cyanobacteria; Evolution;
Titration of E. coli transhydrogenase domain III with bound NADP+ or NADPH studied by NMR reveals no pH-dependent conformational change in the physiological pH range by Anders Pedersen; Tomas Johansson; Jan Rydström; B. Göran Karlsson (254-258).
A pH-titration 2D NMR study of Escherichia coli transhydrogenase domain III with bound NADP+ or NADPH has been carried out, in which the pH was varied between 5.4 and 12. In this analysis, individual amide protons served as reporter groups. The apparent pK a values of the amide protons, determined from the pH-dependent chemical shift changes, were attributed to actual pK a values for several titrating residues in the protein. The essential Asp392 is shown to be protonated at neutral pH in both the NADP+ and NADPH forms of domain III, but with a marked difference in pK a not only attributable to the charge difference between the substrates. Titrating residues found in loop D/α5 point to a conformational difference of these structural elements that is redox-dependent, but not pH dependent. The observed apparent pK a values of these residues are discussed in relation to the crystal structure of Rhodospirillum rubrum domain III, the solution structure of E. coli domain III and the mechanism of intact proton-translocating transhydrogenase.
Keywords: Proton-translocating transhydrogenase; NADP(H); pH-titration; NMR; Domain III; Conformational change;
Cumulative Contents (259-260).