BBA - Bioenergetics (v.1777, #3)
Editorial Board (ii).
Mutations in cytochrome b that affect kinetics of the electron transfer reactions at center N in the yeast cytochrome bc 1 complex by Frederik A.J. Rotsaert; Raul Covian; Bernard L. Trumpower (239-249).
We have examined the pre-steady-state kinetics and thermodynamic properties of the b hemes in variants of the yeast cytochrome bc 1 complex that have mutations in the quinone reductase site (center N). Trp-30 is a highly conserved residue, forming a hydrogen bond with the propionate on the high potential b heme (b H heme). The substitution by a cysteine (W30C) lowers the redox potential of the heme and an apparent consequence is a lower rate of electron transfer between quinol and heme at center N. Leu-198 is also in close proximity to the b H heme and a L198F mutation alters the spectral properties of the heme but has only minor effects on its redox properties or the electron transfer kinetics at center N. Substitution of Met-221 by glutamine or glutamate results in the loss of a hydrophobic interaction that stabilizes the quinone ligands. Ser-20 and Gln-22 form a hydrogen-bonding network that includes His-202, one of the carbonyl groups of the ubiquinone ring, and an active-site water. A S20T mutation has long-range structural effects on center P and thermodynamic effects on both b hemes. The other mutations (M221E, M221Q, Q22E and Q22T) do not affect the ubiquinol oxidation kinetics at center P, but do modify the electron transfer reactions at center N to various extents. The pre-steady reduction kinetics suggest that these mutations alter the binding of quinone ligands at center N, possibly by widening the binding pocket and thus increasing the distance between the substrate and the b H heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function.
Keywords: bc 1, complex; Cytochrome b; Kinetics; Protonmotive Q cycle; Yeast;
Electron transfer kinetics between soluble modules of Paracoccus denitrificans cytochrome c 1 and its physiological redox partners by Julia Janzon; Anna Carina Eichhorn; Bernd Ludwig; Francesco Malatesta (250-259).
The transient electron transfer (ET) interactions between cytochrome c 1 of the bc 1-complex from Paracoccus denitrificans and its physiological redox partners cytochrome c 552 and cytochrome c 550 have been characterized functionally by stopped-flow spectroscopy. Two different soluble fragments of cytochrome c 1 were generated and used together with a soluble cytochrome c 552 module as a model system for interprotein ET reactions. Both c 1 fragments lack the membrane anchor; the c 1 core fragment (c 1CF) consists of only the hydrophilic heme-carrying domain, whereas the c 1 acidic fragment (c 1AF) additionally contains the acidic domain unique to P. denitrificans. In order to determine the ionic strength dependencies of the ET rate constants, an optimized stopped-flow protocol was developed to overcome problems of spectral overlap, heme autoxidation and the prevalent non-pseudo first order conditions. Cytochrome c 1 reveals fast bimolecular rate constants (107 to 108 M− 1 s− 1) for the ET reaction with its physiological substrates c 552 and c 550, thus approaching the limit of a diffusion-controlled process, with 2 to 3 effective charges of opposite sign contributing to these interactions. No direct involvement of the N-terminal acidic c 1-domain in electrostatically attracting its substrates could be detected. However, a slight preference for cytochrome c 550 over c 552 reacting with cyochrome c 1 was found and attributed to the different functions of both cytochromes in the respiratory chain of P. denitrificans.
Keywords: Cytochrome bc 1; Cytochrome c 1 soluble modules; Electron transfer; Paracoccus denitrificans;
Functional characterization of human duodenal cytochrome b (Cybrd1): Redox properties in relation to iron and ascorbate metabolism by Jonathan S. Oakhill; Sophie J. Marritt; Elena Garcia Gareta; Richard Cammack; Andrew T. McKie (260-268).
Duodenal cytochrome b (Dcytb or Cybrd1) is an iron-regulated protein, highly expressed in the duodenal brush border membrane. It has ferric reductase activity and is believed to play a physiological role in dietary iron absorption. Its sequence identifies it as a member of the cytochrome b 561 family. A His-tagged construct of human Dcytb was expressed in insect Sf9 cells and purified. Yields of protein were increased by supplementation of the cells with 5-aminolevulinic acid to stimulate heme biosynthesis. Quantitative analysis of the recombinant Dcytb indicated two heme groups per monomer. Site-directed mutagenesis of any of the four conserved histidine residues (His 50, 86, 120 and 159) to alanine resulted in much diminished levels of heme in the purified Dcytb, while mutation of the non-conserved histidine 33 had no effect on the heme content. This indicates that those conserved histidines are heme ligands, and that the protein cannot stably bind heme if any of them is absent. Recombinant Dcytb was reduced by ascorbate under anaerobic conditions, the extent of reduction being 67% of that produced by dithionite. It was readily reoxidized by ferricyanide. EPR spectroscopy showed signals from low-spin ferriheme, consistent with bis-histidine coordination. These comprised a signal at g max = 3.7 corresponding to a highly anisotropic species, and another at g max = 3.18; these species are similar to those observed in other cytochromes of the b 561 family, and were reducible by ascorbate. In addition another signal was observed in some preparations at g max = 2.95, but this was unreactive with ascorbate. Redox titrations indicated an average midpoint potential for the hemes in Dcytb of + 80 mV ± 30 mV; the data are consistent with either two hemes at the same potential, or differing in potential by up to 60 mV. These results indicate that Dcytb is similar to the ascorbate-reducible cytochrome b 561 of the adrenal chromaffin granule, though with some differences in midpoint potentials of the hemes.
Keywords: Duodenal cytochrome b (Dcytb); Iron; Heme coordination; Electron paramagnetic resonance spectroscopy (EPR spectroscopy); Magnetic circular dichroism-compatible optically-transparent thin-layer electrochemistry (MOTTLE);
Alternative photosynthetic electron flow to oxygen in marine Synechococcus by Shaun Bailey; Anastasios Melis; Katherine R.M. Mackey; Pierre Cardol; Giovanni Finazzi; Gert van Dijken; Gry Mine Berg; Kevin Arrigo; Jeff Shrager; Arthur Grossman (269-276).
Cyanobacteria dominate the world's oceans where iron is often barely detectable. One manifestation of low iron adaptation in the oligotrophic marine environment is a decrease in levels of iron-rich photosynthetic components, including the reaction center of photosystem I and the cytochrome b6f complex [R.F. Strzepek and P.J. Harrison, Photosynthetic architecture differs in coastal and oceanic diatoms, Nature 431 (2004) 689–692.]. These thylakoid membrane components have well characterised roles in linear and cyclic photosynthetic electron transport and their low abundance creates potential impediments to photosynthetic function. Here we show that the marine cyanobacterium Synechococcus WH8102 exhibits significant alternative electron flow to O2, a potential adaptation to the low iron environment in oligotrophic oceans. This alternative electron flow appears to extract electrons from the intersystem electron transport chain, prior to photosystem I. Inhibitor studies demonstrate that a propyl gallate-sensitive oxidase mediates this flow of electrons to oxygen, which in turn alleviates excessive photosystem II excitation pressure that can often occur even at relatively low irradiance. These findings are also discussed in the context of satisfying the energetic requirements of the cell when photosystem I abundance is low.
Keywords: Cyanobacteria; Photosystem; Oxidase; Iron; Oxygen; Electron transport; Alternative electron transport; Synechococcus WH80102;
Electrostatic control of proton pumping in cytochrome c oxidase by Elisa Fadda; Ching-Hsing Yu; Régis Pomès (277-284).
As part of the mitochondrial respiratory chain, cytochrome c oxidase utilizes the energy produced by the reduction of O2 to water to fuel vectorial proton transport. The mechanism coupling proton pumping to redox chemistry is unknown. Recent advances have provided evidence that each of the four observable transitions in the complex catalytic cycle consists of a similar sequence of events. However, the physico-chemical basis underlying this recurring sequence has not been identified. We identify this recurring pattern based on a comprehensive model of the catalytic cycle derived from the analysis of oxygen chemistry and available experimental evidence. The catalytic cycle involves the periodic repetition of a sequence of three states differing in the spatial distribution of charge in the active site: [0|1], [1|0], and [1|1], where the total charge of heme a and the binuclear center appears on the left and on the right, respectively. This sequence recurs four times per turnover despite differences in the redox chemistry. This model leads to a simple, robust, and reproducible sequence of electron and proton transfer steps and rationalizes the pumping mechanism in terms of electrostatic coupling of proton translocation to redox chemistry. Continuum electrostatic calculations support the proposed mechanism and suggest an electrostatic origin for the decoupled and inactive phenotypes of ionic mutants in the principal proton-uptake pathway.
Keywords: Cytochrome c oxidase; Vectorial proton transport; Redox-coupled proton transport; Proton-pumping mechanism; Binuclear center; Continuum electrostatics;
Spin-probes designed for measuring the intrathylakoid pH in chloroplasts by Alexander N. Tikhonov; Roman V. Agafonov; Igor A. Grigor'ev; Igor A. Kirilyuk; Vasilii V. Ptushenko; Boris V. Trubitsin (285-294).
Nitroxide radicals are widely used as molecular probes in different fields of chemistry and biology. In this work, we describe pH-sensitive imidazoline- and imidazolidine-based nitroxides with pK values in the range 4.7–7.6 (2,2,3,4,5,5-hexamethylperhydroimidazol-1-oxyl, 4-amino-2,2,5,5-tetramethyl-2,5-dihydro-1H-imidazol-1-oxyl, 4-dimethylamino-2,2-diethyl-5,5-dimethyl-2,5-dihydro-1H-imidazol-1-oxyl, and 2,2-diethyl-5,5-dimethyl-4-pyrrolidyline-1-yl-2,5-dihydro-1H-imidazol-1-oxyl), which allow the pH-monitoring inside chloroplasts. We have demonstrated that EPR spectra of these spin-probes localized in the thylakoid lumen markedly change with the light-induced acidification of the thylakoid lumen in chloroplasts. Comparing EPR spectrum parameters of intrathylakoid spin-probes with relevant calibrating curves, we could estimate steady-state values of lumen pHin established during illumination of chloroplasts with continuous light. For isolated bean (Vicia faba) chloroplasts suspended in a medium with pHout = 7.8, we found that pHin ≈ 5.4–5.7 in the state of photosynthetic control, and pHin ≈ 5.7–6.0 under photophosphorylation conditions. Thus, ATP synthesis occurs at a moderate acidification of the thylakoid lumen, corresponding to transthylakoid pH difference ΔpH ≈ 1.8–2.1. These values of ΔpH are consistent with a point of view that under steady-state conditions the proton gradient ΔpH is the main contributor to the proton motive force driving the operation of ATP synthesis, provided that stoichiometric ratio H+/ATP is n ≥ 4–4.7.
Keywords: Chloroplast; Electron paramagnetic resonance; Spin-probes; Transthylakoid proton gradient;
Pulse ENDOR and density functional theory on the peridinin triplet state involved in the photo-protective mechanism in the peridinin–chlorophyll a–protein from Amphidinium carterae by Marilena Di Valentin; Stefano Ceola; Giancarlo Agostini; Giorgio Mario Giacometti; Alexander Angerhofer; Orlando Crescenzi; Vincenzo Barone; Donatella Carbonera (295-307).
The photoexcited triplet state of the carotenoid peridinin in the Peridinin–chlorophyll a–protein of the dinoflagellate Amphidinium carterae has been investigated by pulse EPR and pulse ENDOR spectroscopies at variable temperatures. This is the first time that the ENDOR spectra of a carotenoid triplet in a naturally occurring light-harvesting complex, populated by energy transfer from the chlorophyll a triplet state, have been reported. From the electron spin echo experiments we have obtained the information on the electron spin polarization dynamics and from Mims ENDOR experiments we have derived the triplet state hyperfine couplings of the α- and β-protons of the peridinin conjugated chain. Assignments of β-protons belonging to two different methyl groups, with a iso = 7.0 MHz and a iso = 10.6 MHz respectively, have been made by comparison with the values predicted from density functional theory. Calculations provide a complete picture of the triplet spin density on the peridinin molecule, showing that the triplet spins are delocalized over the whole π-conjugated system with an alternate pattern, which is lost in the central region of the polyene chain. The ENDOR investigation strongly supports the hypothesis of localization of the triplet state on one peridinin in each subcluster of the PCP complex, as proposed in [Di Valentin et al. Biochim. Biophys. Acta 1777 (2008) 186–195]. High spin density has been found specifically at the carbon atom at position 12 (see Fig. 1B), which for the peridinin involved in the photo-protective mechanism is in close contact with the water ligand to the chlorophyll a pigment. We suggest that this ligated water molecule, placed at the interface between the chlorophyll–peridinin pair, is functioning as a bridge in the triplet–triplet energy transfer between the two pigments.
Keywords: PCP; Carotenoid; Triplet state; Pulse EPR; Pulse ENDOR; DFT; Hyperfine couplings;
The PsaE subunit of photosystem I prevents light-induced formation of reduced oxygen species in the cyanobacterium Synechocystis sp. PCC 6803 by Robert Jeanjean; Amel Latifi; Hans C.P. Matthijs; Michel Havaux (308-316).
The PsaE protein is located at the reducing side of photosystem I (PSI) and is involved in docking the soluble electron acceptors, particularly ferredoxin. However, deletion of the psaE gene in the cyanobacterium Synechocystis sp. strain PCC 6803 inhibited neither photoautotrophic growth, nor in vivo linear and cyclic electron flows. Using photoacoustic spectroscopy, we detected an oxygen-dependent, PSI-mediated energy storage activity in the ΔpsaE null mutant, which was not present in the wild type (WT). The expression of the genes encoding catalase (katG) and iron superoxide dismutase (sodB) was upregulated in the ΔpsaE mutant, and the increase in katG expression was correlated with an increase in catalase activity of the cells. When catalases were inhibited by sodium azide, the production of reactive oxygen species was enhanced in ΔpsaE relative to WT. Moreover, sodium azide strongly impaired photoautotrophic growth of the ΔpsaE mutant cells while WT was much less sensitive to this inhibitor. The katG gene was deleted in the ΔpsaE mutant, and the resulting double mutant was more photosensitive than the single mutants, showing cell bleaching and lipid peroxidation in high light. Our results show that the presence of the PsaE polypeptide at the reducing side of PSI has a function in avoidance of electron leakage to oxygen in the light (Mehler reaction) and the resulting formation of toxic oxygen species. PsaE-deficient Synechocystis cells can counteract the chronic photoreduction of oxygen by increasing their capacity to detoxify reactive oxygen species.
Keywords: Photosystem; Photooxidative stress; Oxygen photoreduction; Catalase; Lipid peroxidation; Cyanobacteria;
Formation of engineered intersubunit disulfide bond in cytochrome bc 1 complex disrupts electron transfer activity in the complex by He-Wen Ma; Shaoqing Yang; Linda Yu; Chang-An Yu (317-326).
Protein domain movement of the Rieske iron–sulfur protein has been speculated to play an essential role in the bifurcated oxidation of ubiquinol catalyzed by the cytochrome bc 1 complex. To better understand the electron transfer mechanism of the bifurcated ubiquinol oxidation at Qp site, we fixed the head domain of ISP at the cyt c 1 position by creating an intersubunit disulfide bond between two genetically engineered cysteine residues: one at position 141 of ISP and the other at position 180 of the cyt c 1 [S141C(ISP)/G180C(cyt c 1)]. The formation of a disulfide bond between ISP and cyt c 1 in this mutant complex is confirmed by SDS-PAGE and Western blot. In this mutant complex, the disulfide bond formation is concurrent with the loss of the electron transfer activity of the complex. When the disulfide bond is released by treatment with β-mercaptoethanol, the activity is restored. These results further support the hypothesis that the mobility of the head domain of ISP is functionally important in the cytochrome bc 1 complex. Formation of the disulfide bond between ISP and cyt c 1 shortens the distance between the [2Fe–2S] cluster and heme c 1, hence the rate of intersubunit electron transfer between these two redox prosthetic groups induced by pH change is increased. The intersubunit disulfide bond formation also decreases the rate of stigmatellin induced reduction of ISP in the fully oxidized complex, suggesting that an endogenous electron donor comes from the vicinity of the b position in the cytochrome b.
Keywords: Bacterial cytochrome bc 1 complex; Formation of a disulfide bond between ISP and cyt c 1; Head domain of ISP; Rhodobacter sphaeroides;