BBA - Bioenergetics (v.1817, #12)
Editorial Board (i).
The dynamics of the non-heme iron in bacterial reaction centers from Rhodobacter sphaeroides by A. Hałas; A. Orzechowska; V. Derrien; A.I. Chumakov; P. Sebban; J. Fiedor; M. Lipińska; M. Zając; T. Ślęzak; K. Strzałka; K. Matlak; J. Korecki; L. Fiedor; K. Burda (2095-2102).
We investigate the dynamical properties of the non-heme iron (NHFe) in His-tagged photosynthetic bacterial reaction centers (RCs) isolated from Rhodobacter (Rb.) sphaeroides. Mössbauer spectroscopy and nuclear inelastic scattering of synchrotron radiation (NIS) were applied to monitor the arrangement and flexibility of the NHFe binding site. In His-tagged RCs, NHFe was stabilized only in a high spin ferrous state. Its hyperfine parameters (IS = 1.06 ± 0.01 mm/s and QS = 2.12 ± 0.01 mm/s), and Debye temperature (θD0 ~ 167 K) are comparable to those detected for the high spin state of NHFe in non-His-tagged RCs. For the first time, pure vibrational modes characteristic of NHFe in a high spin ferrous state are revealed. The vibrational density of states (DOS) shows some maxima between 22 and 33 meV, 33 and 42 meV, and 53 and 60 meV and a very sharp one at 44.5 meV. In addition, we observe a large contribution of vibrational modes at low energies. This iron atom is directly connected to the protein matrix via all its ligands, and it is therefore extremely sensitive to the collective motions of the RC protein core. A comparison of the DOS spectra of His-tagged and non-His-tagged RCs from Rb. sphaeroides shows that in the latter case the spectrum was overlapped by the vibrations of the heme iron of residual cytochrome c2, and a low spin state of NHFe in addition to its high spin one. This enabled us to pin-point vibrations characteristic for the low spin state of NHFe.► Only high spin NHFe occurs in His-tagged bacterial RCs. ► Its hyperfine parameters and flexibility are similar to NHFe in non-His-tagged RCs. ► Pure vibrational modes characteristic of NHFe are observed. ► Some vibrations sensitive to different spin states of NHFe are indicated.
Keywords: Photosynthetic reaction center; Non-heme iron; Mössbauer spectroscopy; Nuclear inelastic scattering;
Oxygen dependent electron transfer in the cytochrome bc 1 complex by Fei Zhou; Ying Yin; Ting Su; Linda Yu; Chang-An Yu (2103-2109).
The effect of molecular oxygen on the electron transfer activity of the cytochrome bc 1 complex was investigated by determining the activity of the complex under the aerobic and anaerobic conditions. Molecular oxygen increases the activity of Rhodobacter sphaeroides bc 1 complex up to 82%, depending on the intactness of the complex. Since oxygen enhances the reduction rate of heme b L, but shows no effect on the reduction rate of heme b H, the effect of oxygen in the electron transfer sequence of the cytochrome bc 1 complex is at the step of heme b L reduction during bifurcated oxidation of ubiquinol.► The cytochrome bc 1 catalyzes the electron transfer from ubiquinol to cytochrome c. ► The activity of cytochrome bc 1 increases in the presence of molecular oxgyen. ► The electron transfer catalyzed by the cytochrome bc 1 is oxygen dependent. ► The effect of oxygen is dependent on the intactness of the complex. ► The effect of oxygen is at the step of heme b L reduction.
Keywords: Cytochrome; Ubiquinone; Oxygen; Superoxide ion; Electron transfer; Bioenergetics;
Proteases are associated with a minor fucoxanthin chlorophyll a/c-binding protein from the diatom, Chaetoceros gracilis by Ryo Nagao; Tatsuya Tomo; Eri Noguchi; Takehiro Suzuki; Akinori Okumura; Rei Narikawa; Isao Enami; Masahiko Ikeuchi (2110-2117).
We previously showed that most subunits in the oxygen-evolving photosystem II (PSII) preparation from the diatom Chaetoceros gracilis are proteolytically unstable. Here, we focused on identifying the proteases that cleave PSII subunits in thylakoid membranes. Major PSII subunits and fucoxanthin chlorophyll (Chl) a/c‐binding proteins (FCPs) were specifically degraded in thylakoid membranes. The PSI subunits, PsaA and PsaB, were slowly degraded, and cytochrome f was barely degraded. Using zymography, proteolytic activities for three metalloproteases (116, 83, and 75 kDa) and one serine protease (156 kDa) were detected in thylakoid membranes. Two FCP fractions (FCP-A and FCP-B/C) and a photosystem fraction were separated by sucrose gradient centrifugation using dodecyl maltoside‐solubilized thylakoids. The FCP-A fraction featured enriched Chl c compared with the bulk of FCP-B/C. Zymography revealed that 116, 83, and 94 kDa metalloproteases were mostly in the FCP-A fraction along with the 156 kDa serine protease. When solubilized thylakoids were separated with clear-native PAGE, zymography detected only the 83 kDa metalloprotease in the FCP-A band. Because FCP-A is selectively associated with PSII, these FCP-A-associated metalloproteases and serine protease may be responsible for the proteolytic degradation of FCPs and PSII in thylakoid membranes.► Proteolysis of PSII and FCPs was observed in PSII and thylakoids from C. gracilis. ► Metalloproteases and serine protease were identified in thylakoids. ► Those proteases were associated with a minor FCP. ► The FCP may play multiple roles in light harvesting and/or protein turnover of PSII.
Keywords: FCP; Photosystem; Protease; Thylakoid;
Understanding the FMN cofactor chemistry within the Anabaena Flavodoxin environment by Isaias Lans; Susana Frago; Milagros Medina (2118-2127).
The chemical versatility of flavin cofactors within the flavoprotein environment allows them to play main roles in the bioenergetics of all type of organisms, particularly in energy transformation processes such as photosynthesis or oxidative phosphorylation. Despite the large diversity of properties shown by flavoproteins and of the biological processes in which they are involved, only two flavin cofactors, FMN and FAD (both derived from the 7,8-dimethyl-10-(1′-D-ribityl)-isoalloxazine), are usually found in these proteins. Using theoretical and experimental approaches we have carried out an evaluation of the effects introduced upon substituting the 7- and/or 8-methyls of the isoalloxazine ring in the chemical and oxido-reduction properties of the different atoms of the ring on free flavins and on the photosynthetic Anabaena Flavodoxin (a flavoprotein that replaces Ferredoxin as electron carrier from Photosystem I to Ferredoxin-NADP+ reductase). In Anabaena Flavodoxin both the protein environment and the redox state contribute to modulate the chemical reactivity of the isoalloxazine ring. Anabaena apoflavodoxin is shown to be designed to stabilise/destabilise each one of the FMN redox states (but not of the analogues produced upon substitution of the 7- and/or 8-methyls groups) in the adequate proportions to provide Flavodoxin with the particular properties required for the functions in which it is involved in vivo. The 7- and/or 8-methyl groups of the ixoalloxazine can be discarded as the gate for electrons exchange in Anabaena Fld, but a key role in this process is envisaged for the C6 atom of the flavin and the backbone atoms of Asn58.► Use of photosynthetic flavodoxin to study flavins chemical reactivity ► Isoalloxazine substitutions on physico-chemical properties of free flavins ► Isoalloxazine substitutions on physico-chemical properties of Flavodoxin ► Apoprotein and redox state modulate the isoalloxazine ring chemical reactivity. ► ApoFld sta/destabilises FMN redox states providing properties for in vivo function.
Keywords: Flavodoxin; Isoalloxazine; FMN analogue; FMN affinity; QM/MM calculation; Chemical reactivity;
The cytosol-synthesized subunit II (Cox2) precursor with the point mutation W56R is correctly processed in yeast mitochondria to rescue cytochrome oxidase by Valentín Cruz-Torres; Miriam Vázquez-Acevedo; Rodolfo García-Villegas; Xochitl Pérez-Martínez; Guillermo Mendoza-Hernández; Diego González-Halphen (2128-2139).
Deletion of the yeast mitochondrial gene COX2 encoding subunit 2 (Cox2) of cytochrome c oxidase (CcO) results in loss of respiration (Δcox2 strain). Supekova et al. (2010)  transformed a Δcox2 strain with a vector expressing Cox2 with a mitochondrial targeting sequence (MTS) and the point mutation W56R (Cox2W56R), restoring respiratory growth. Here, the CcO carrying the allotopically-expressed Cox2W56R was characterized. Yeast mitochondria from the wild-type (WT) and the Δcox2 + Cox2W56R strains were subjected to Blue Native electrophoresis. In-gel activity of CcO and spectroscopic quantitation of cytochromes revealed that only 60% of CcO is present in the complemented strain, and that less CcO is found associated in supercomplexes as compared to WT. CcOs from the WT and the mutant exhibited similar subunit composition, although activity was 20–25% lower in the enzyme containing Cox2W56R than in the one with Cox2WT. Tandem mass spectrometry confirmed that W56 was substituted by R56 in Cox2W56R. In addition, Cox2W56R exhibited the same N-terminus than Cox2WT, indicating that the MTS of Oxa1 and the leader sequence of 15 residues were removed from Cox2W56R during maturation. Thus, Cox2W56R is identical to Cox2WT except for the point mutation W56R. Mitochondrial Cox1 synthesis is strongly reduced in Δcox2 mutants, but the Cox2W56R complemented strain led to full restoration of Cox1 synthesis. We conclude that the cytosol-synthesized Cox2W56R follows a rate-limiting process of import, maturation or assembly that yields lower steady-state levels of CcO. Still, the allotopically-expressed Cox2W56R restores CcO activity and allows mitochondrial Cox1 synthesis to advance at WT levels.Display Omitted► A cytosol-synthesized Cox2W56R precursor is correctly assembled into cytochrome oxidase (CcO). ► CcO carrying Cox2W56R accumulates 40% less than in the WT strain. ► CcO carrying Cox2W56R also accumulates less supercomplexes than the WT strain. ► The presence of Cox2W56R fully restored mitochondrial Cox1 synthesis in the Δcox2 mutant. ► This is the first characterization of a CcO carrying an allotopically-expressed subunit with two TMS.
Keywords: Allotopic expression; Mitochondrial targeting signals; Subunit 2 of cytochrome c oxidase; Cytochrome c oxidase biogenesis;
Kinetic properties and physiological role of the plastoquinone terminal oxidase (PTOX) in a vascular plant by Martin Trouillard; Maryam Shahbazi; Lucas Moyet; Fabrice Rappaport; Pierre Joliot; Marcel Kuntz; Giovanni Finazzi (2140-2148).
The physiological role of the plastid terminal oxidase (PTOX) involved in plastoquinol oxidation in chloroplasts has been investigated in vivo in tomato leaves. Enzyme activity was assessed by non-invasive methods based on the analysis of the kinetics of chlorophyll fluorescence changes. In the dark, the maximum PTOX rate was smaller than 1 electron per second per PSII. This value was further decreased upon light acclimation, and became almost negligible upon inhibition of the photosynthetic performances by reducing the CO2 availability. In contrast, prolonged exposure to high light resulted in an increase of the overall PTOX activity, which was paralleled by an increased protein accumulation. Under all the conditions tested the enzyme activity always remained about two orders of magnitude lower than that of electron flux through the linear photosynthetic electron pathway. Therefore, PTOX cannot have a role of a safety valve for photogenerated electrons, while it could be involved in acclimation to high light. Moreover, by playing a major role in the control of the stromal redox poise, PTOX is also capable of modulating the balance between linear and cyclic electron flow around PSI during the deactivation phase of carbon assimilation that follows a light to dark transition.► The chlororespiratory terminal oxidase (PTOX) activity was assessed by non-invasive methods. ► PTOX activity is not compatible with a flux expected for an effective photosynthetic electron sink. ► PTOX influences the transition from linear to cyclic electron flow during a dark recovery.
Keywords: Plastid terminal oxidase; Plastoquinone pool; Photosystem; NADPH dehydrogenase; Cyclic electron transfer;
The [4Fe–4S]-cluster coordination of [FeFe]-hydrogenase maturation protein HydF as revealed by EPR and HYSCORE spectroscopies by Paola Berto; Marilena Di Valentin; Laura Cendron; Francesca Vallese; Marco Albertini; Enrico Salvadori; Giorgio M. Giacometti; Donatella Carbonera; Paola Costantini (2149-2157).
[FeFe] hydrogenases are key enzymes for bio(photo)production of molecular hydrogen, and several efforts are underway to understand how their complex active site is assembled. This site contains a [4Fe–4S]-2Fe cluster and three conserved maturation proteins are required for its biosynthesis. Among them, HydF has a double task of scaffold, in which the dinuclear iron precursor is chemically modified by the two other maturases, and carrier to transfer this unit to a hydrogenase containing a preformed [4Fe–4S]-cluster. This dual role is associated with the capability of HydF to bind and dissociate an iron–sulfur center, due to the presence of the conserved FeS-cluster binding sequence CxHx46–53HCxxC. The recently solved three-dimensional structure of HydF from Thermotoga neapolitana described the domain containing the three cysteines which are supposed to bind the FeS cluster, and identified the position of two conserved histidines which could provide the fourth iron ligand. The functional role of two of these cysteines in the activation of [FeFe]-hydrogenases has been confirmed by site-specific mutagenesis. On the other hand, the contribution of the three cysteines to the FeS cluster coordination sphere is still to be demonstrated. Furthermore, the potential role of the two histidines in [FeFe]-hydrogenase maturation has never been addressed, and their involvement as fourth ligand for the cluster coordination is controversial. In this work we combined site-specific mutagenesis with EPR (electron paramagnetic resonance) and HYSCORE (hyperfine sublevel correlation spectroscopy) to assign a role to these conserved residues, in both cluster coordination and hydrogenase maturation/activation, in HydF proteins from different microorganisms.► Alternative non-cysteinyl FeS cluster coordination exists in different HydF proteins. ► Three conserved cysteines are strictly required for HydF FeS cluster ligation. ► HydF conserved histidines can be substituted by other ligands in iron coordination. ► Structural flexibility characterizes the FeS cluster binding site of HydF.
Keywords: Iron–sulfur proteins; Hydrogenase; HydF; Iron–sulfur cluster coordination; EPR;
The role of the PsbS protein in the protection of photosystems I and II against high light in Arabidopsis thaliana by Thomas Roach; Anja Krieger-Liszkay (2158-2165).
The PsbS protein is recognised in higher plants as an important component in dissipating excess light energy via its regulation of non-photochemical quenching. We investigated photosynthetic responses in the arabidopsis npq4 mutant, which lacks PsbS, and in a mutant over-expressing PsbS (oePsbS). Growth under low light led to npq4 and wild-type plants being visibly indistinguishable, but induced a phenotype in oePsbS plants, which were smaller and had shorter flowering spikes. Here we report that chloroplasts from npq4 generated more singlet oxygen (1O2) than those from oePsbS. This accompanied a higher extent of photosystem II photoinhibition of leaves from npq4 plants. In contrast, oePsbS was more damaged by high light than npq4 and the wild-type at the level of photosystem I. The plastoquinone pool, as measured by thermoluminescence, was more oxidised in the oePsbS than in npq4, whilst the amount of photo-oxidisable P700, as probed with actinic light or saturating flashes, was higher in oePsbS compared to wild-type and npq4. Taken together, this indicates that the level of PsbS has a regulatory role in cyclic electron flow. Overall, we show that under high light oePsbS plants were more protected from 1O2 at the level of photosystem II, whereas lack of cyclic electron flow rendered them susceptible to damage at photosystem I. Cyclic electron flow is concluded to be essential for protecting photosystem I from high light stress.Phenotypes of the low light-grown arabidopsis.Display Omitted► Chloroplasts from npq4 arabidopsis produce more singlet oxygen than wild-type. ► PSII of npq4 is more susceptible to high light than wild-type. ► The plastoquinone redox state is related to the amount of PsbS. ► Cyclic electron flow is inhibited in PsbS over-expressing plants. ► PSI of PsbS over-expressing plants is more sensitive to high light than npq4.
Keywords: Singlet oxygen; Photosynthesis; Non-photochemical quenching; Cyclic electron flow; Photoinhibition; Arabidopsis;