BBA - Bioenergetics (v.1827, #3)
Editorial Board (i).
Electrochemical behaviour of bacterial nitric oxide reductase—Evidence of low redox potential non-heme FeB gives new perspectives on the catalytic mechanism by Cristina M. Cordas; Américo G. Duarte; José J.G. Moura; Isabel Moura (233-238).
Nitric oxide reductase (NOR) is a membrane bound enzyme involved in the metabolic denitrification pathway, reducing nitric oxide (NO) to nitrous oxide (N2O), subsequently promoting the formation of the N―N bond. Three types of bacterial NOR are known, namely cNOR, qNOR and qCuNOR, that differ on the physiological electron donor. cNOR has been purified as a two subunit complex, the NorC, anchored to the cytoplasmic membrane, with a low-spin heme c, and the NorB subunit showing high structural homology with the HCuO subunit I, comprising a bis-histidine low-spin heme b and a binuclear iron centre. The binuclear iron centre is the catalytic site and it is formed by a heme b 3 coupled to a non-heme iron (FeB) through a μ-oxo bridge. The catalytic mechanism is still under discussion and three hypotheses have been proposed: the trans-mechanism, the cis-FeB and the cis-heme b 3 mechanisms. In the present work, the Pseudomonas nautica cNOR electrochemical behaviour was studied by cyclic voltammetry (CV), using a pyrolytic graphite electrode modified with the immobilised protein. The protein redox centres were observed and the formal redox potentials were determined. The binuclear iron centre presents the lowest redox potential value, and discrimination between the heme b 3 and FeB redox processes was attained. Also, the number of electrons involved and correspondent surface electronic transfer rate constants were estimated. The pH dependence of the observed redox processes was determined and some new insights on the NOR catalytic mechanism are discussed.► This work reports the cNOR characterization attained via direct electron transfer. ► Metal centre midpoint potentials and number of involved electrons were estimated. ► Redox centres' pH dependence was evaluated. ► Non-heme iron FeB process occurs at lower potential value than previously reported. ► Electrochemical results of heme b 3 on turnover conditions point to cis-FeB mechanism.
Keywords: Denitrification; Electrochemistry; Enzymatic catalysis; Metalloenzyme; Nitric oxide reductase;
Modulation of mitochondrial activity by S-nitrosoglutathione reductase in Arabidopsis thaliana transgenic cell lines by Lucas Frungillo; Jusceley Fatima Palamim de Oliveira; Elzira Elisabeth Saviani; Halley Caixeta Oliveira; M. Carmen Martínez; Ione Salgado (239-247).
The enzyme S-nitrosoglutathione reductase (GSNOR) has an important role in the metabolism of S-nitrosothiols (SNO) and, consequently, in the modulation of nitric oxide (NO)-mediated processes. Although the mitochondrial electron transport chain is an important target of NO, the role of GSNOR in the functionality of plant mitochondria has not been addressed. Here, we measured SNO content and NO emission in Arabidopsis thaliana cell suspension cultures of wild-type (WT) and GSNOR overexpressing (GSNOROE) or antisense (GSNORAS) transgenic lines, grown under optimal conditions and under nutritional stress. Respiratory activity of isolated mitochondria and expression of genes encoding for mitochondrial proteins were also analyzed. Under optimal growth conditions, GSNOROE had the lowest SNO and NO levels and GSNORAS the highest, as expected by the GSNO-consuming activity of GSNOR. Under stress, this pattern was reversed. Analysis of oxygen uptake by isolated mitochondria showed that complex I and external NADH dehydrogenase activities were inhibited in GSNOROE cells grown under nutritional stress. Moreover, GSNOROE could not increase alternative oxidase (AOX) activity under nutritional stress. GSNORAS showed constitutively high activity of external NADH dehydrogenase, and maintained the activity of the uncoupling protein (UCP) under stress. The alterations observed in mitochondrial protein activities were not strictly correlated to changes in gene expression, but instead seemed to be related with the SNO/NO content, suggesting a post-transcriptional regulation. Overall, our results highlight the importance of GSNOR in modulating SNO and NO homeostasis as well mitochondrial functionality, both under normal and adverse conditions in A. thaliana cells.► GSNOR modulates mitochondrial respiratory activity and energy conservation. ► Activities of plant mitochondrial respiratory proteins are responsive to GSNOR. ► SNO/NO content regulates mitochondrial respiratory activity.
Keywords: GSNO reductase; Nitric oxide; Alternative oxidase; Uncoupling protein; Respiratory chain; Arabidopsis thaliana;
Carotenoid–protein interaction alters the S1 energy of hydroxyechinenone in the Orange Carotenoid Protein by Tomáš Polívka; Pavel Chábera; Cheryl A. Kerfeld (248-254).
The Orange Carotenoid Protein (OCP) is a photoactive water soluble protein that is crucial for photoprotection in cyanobacteria. When activated by blue-green light, it triggers quenching of phycobilisome fluorescence and regulates energy flow from the phycobilisome to the reaction center. The OCP contains a single pigment, the carotenoid 3′-hydroxyechinenone (hECN). Binding to the OCP causes a conformational change in hECN leading to an extension of its effective conjugation length. We have determined the S1 energy of hECN in organic solvent and compared it with the S1 energy of hECN bound to the OCP. In methanol and n-hexane, hECN has an S1 energy of 14,300 cm− 1, slightly higher than carotenoids with shorter conjugation lengths such as zeaxanthin or β-carotene; this is consistent with the proposal that the presence of the conjugated carbonyl group in hECN increases its S1 energy. The S1 energy of hECN in organic solvent is independent of solvent polarity. Upon binding to the OCP, the S1 energy of hECN is further increased to 14,700 cm− 1, underscoring the importance of protein binding which twists the conjugated carbonyl group into s-trans conformation and enhances the effect of the carbonyl group. Activated OCP, however, has an S1 energy of 14,000 cm− 1, indicating that significant changes in the vicinity of the conjugated carbonyl group occur upon activation.► Active and inactive forms of OCP coexist in OCP sample at daylight. ► Hydroxyechinenone S1 energy in inactive OCP is higher than in solution. ► Activation of OCP lowers the S1 energy of hydroxyechinenone bound to OCP.
Keywords: Photoprotection; Cyanobacteria; Carotenoid; Orange Carotenoid Protein; Femtosecond transient absorption spectroscopy;
Photosystem trap energies and spectrally-dependent energy-storage efficiencies in the Chl d-utilizing cyanobacterium, Acaryochloris marina by Steven P. Mielke; Nancy Y. Kiang; Robert E. Blankenship; David Mauzerall (255-265).
Acaryochloris marina is the only species known to utilize chlorophyll (Chl) d as a principal photopigment. The peak absorption wavelength of Chl d is redshifted ≈ 40 nm in vivo relative to Chl a, enabling this cyanobacterium to perform oxygenic phototrophy in niche environments enhanced in far-red light. We present measurements of the in vivo energy-storage (E-S) efficiency of photosynthesis in A. marina, obtained using pulsed photoacoustics (PA) over a 90-nm range of excitation wavelengths in the red and far-red. Together with modeling results, these measurements provide the first direct observation of the trap energies of PSI and PSII, and also the photosystem-specific contributions to the total E-S efficiency. We find the maximum observed efficiency in A. marina (40 ± 1% at 735 nm) is higher than in the Chl a cyanobacterium Synechococcus leopoliensis (35 ± 1% at 690 nm). The efficiency at peak absorption wavelength is also higher in A. marina (36 ± 1% at 710 nm vs. 31 ± 1% at 670 nm). In both species, the trap efficiencies are ≈ 40% (PSI) and ≈ 30% (PSII). The PSI trap in A. marina is found to lie at 740 ± 5 nm, in agreement with the value inferred from spectroscopic methods. The best fit of the model to the PA data identifies the PSII trap at 723 ± 3 nm, supporting the view that the primary electron-donor is Chl d, probably at the accessory (ChlD1) site. A decrease in efficiency beyond the trap wavelength, consistent with uphill energy transfer, is clearly observed and fit by the model. These results demonstrate that the E-S efficiency in A. marina is not thermodynamically limited, suggesting that oxygenic photosynthesis is viable in even redder light environments.► Photoacoustic measurements of energy-storage efficiency over a 90-nm spectral range in A. marina ► First direct observation of the trap energies of PSI and PSII ► Photosystem-specific contributions to the observed efficiency ► Maximum observed efficiency in A. marina (40 ± 1%) is higher than in S. leopoliensis (35 ± 1%). ► PSII trap in A. marina at 723 nm supports evidence that primary donor is Chl d at ChlD1 site.
Keywords: Acaryochloris marina; Chlorophyll d; Photosynthetic energy-storage; Photosynthetic efficiency; Limits of oxygenic photosynthesis; Photoacoustics;
Characterization of the Type III sulfide:quinone oxidoreductase from Caldivirga maquilingensis and its membrane binding by Andrea M. Lencina; Ziqiao Ding; Lici A. Schurig-Briccio; Robert B. Gennis (266-275).
Sulfide:quinone oxidoreductases (SQRs) are ubiquitous enzymes which have multiple roles: sulfide detoxification, energy generation by providing electrons to respiratory or photosynthetic electron transfer chains, and sulfide homeostasis. A recent structure-based classification defines 6 groups of putative SQRs (I–VI), and representatives of all but group III have been confirmed to have sulfide oxidase activity. In the current work, we report the first characterization of a predicted group III SQR from Caldivirga maquilingensis, and confirm that this protein is a sulfide oxidase. The gene encoding the enzyme was cloned, and the protein was expressed in E. coli and purified. The enzyme oxidizes sulfide using decylubiquinone as an electron acceptor, and is inhibited by aurachin C and iodoacetamide. Analysis of the amino acid sequence indicates that the C. maquilingensis SQR has two amphiphilic helices at the C-terminus but lacks any transmembrane helices. This suggests that C. maquilingensis SQR interacts with the membrane surface and that the interactions are mediated by the C-terminal amphiphilic helices. Mutations within the last C-terminal amphiphilic helix resulted in a water-soluble form of the enzyme which, remarkably, retains full SQR activity using decylubiquinone as the electron acceptor. Mutations at one position, L379, also located in the C-terminal amphiphilic helix, inactivated the enzyme by preventing the interaction with decylubiquinone. It is concluded that the C-terminal amphiphilic helix is important for membrane binding and for forming part of the pathway providing access of the quinone substrate to the protein-bound flavin at the enzyme active site.► The sulfide:quinone oxidoreductase (SQR) from C. maquilingensis was characterized. ► This is the first representative of a “Class III” SQR to be characterized. ► Membrane binding is mediated by residues in the C-terminal amphiphilic helix. ► Mutations in this portion of the protein convert the enzyme to a soluble form.
Keywords: Sulfide:quinone oxidoreductase; Caldivirga maquilingensis; Monotopic membrane protein; Amphiphilic helix;
Exploring the entrance of proton pathways in cytochrome c oxidase from Paracoccus denitrificans: Surface charge, buffer capacity and redox-dependent polarity changes at the internal surface by Kristina Kirchberg; Hartmut Michel; Ulrike Alexiev (276-284).
Cytochrome c oxidase (CcO), the terminal oxidase of cellular respiration, reduces molecular oxygen to water. The mechanism of proton pumping as well as the coupling of proton and electron transfer is still not understood in this redox-linked proton pump. Eleven residues at the aqueous-exposed surfaces of CcO from Paracoccus denitrificans have been exchanged to cysteines in a two-subunit base variant to yield single reactive cysteine variants. These variants are designed to provide unique labeling sites for probes to be used in spectroscopic experiments investigating the mechanism of proton pumping in CcO. To this end we have shown that all cysteine variants are enzymatically active. Cysteine positions at the negative (N-) side of the membrane are located close to the entrance of the D- and K-proton transfer pathways that connect the N-side with the catalytic oxygen reduction site. Labeling of the pH-indicator dye fluorescein to these sites allowed us to determine the surface potential at the cytoplasmic CcO surface, which corresponds to a surface charge density of − 0.5 elementary charge/1000 Å2. In addition, acid–base titrations revealed values of CcO buffer capacity. Polarity measurements of the label environment at the N-side provided (i) site-specific values indicative of a hydrophilic and a more hydrophobic environment dependent on the label position, and (ii) information on a global change to a more apolar environment upon reduction of the enzyme. Thus, the redox state of the copper and heme centers inside the hydrophobic interior of CcO affect the properties at the cytoplasmic surface.► Eleven single-reactive cysteine variants of cytochrome c oxidase (CcO) from P. denitrificans were constructed. ► Modification of the cysteine residues with the fluorescent dye fluorescein was demonstrated. ► A distinct polarity pattern for the fluorescein attachment sites at the entrance of the D- and K-channel of CcO was observed. ► The cytoplasmic surface of CcO becomes more apolar upon reduction of the enzyme.
Keywords: Cytochrome c oxidase; pH-indicator dye; Covalent surface labeling; Surface potential; Buffer capacity; Fluorescein;
LYRM7/MZM1L is a UQCRFS1 chaperone involved in the last steps of mitochondrial Complex III assembly in human cells by Ester Sánchez; Teresa Lobo; Jennifer L. Fox; Massimo Zeviani; Dennis R. Winge; Erika Fernández-Vizarra (285-293).
The mammalian Complex III (CIII) assembly process is yet to be completely understood. There is still a lack in understanding of how the structural subunits are put together and which additional factors are involved. Here we describe the identification and characterization of LYRM7, a human protein displaying high sequence homology to the Saccharomyces cerevisiae protein Mzm1, which was recently shown as an assembly factor for Rieske Fe–S protein incorporation into the yeast cytochrome bc1 complex. We conclude that human LYRM7, which we propose to be renamed MZM1L (MZM1-like), works as a human Rieske Fe–S protein (UQCRFS1) chaperone, binding to this subunit within the mitochondrial matrix and stabilizing it prior to its translocation and insertion into the late CIII dimeric intermediate within the mitochondrial inner membrane. Thus, LYRM7/MZM1L is a novel human CIII assembly factor involved in the UQCRFS1 insertion step, which enables formation of the mature and functional CIII enzyme.► Human LYRM7 is identified as the yeast Mzm1 functional homolog ► LYRM7/MZM1L is a new human Complex III assembly factor ► MZM1L is a UQCRFS1 chaperone, binding and stabilizing it prior to its translocation ► Correct insertion of UQCRFS1 to mature Complex III depends on MZM1L stoichiometry
Keywords: Mitochondrial respiratory chain; Complex III; Assembly factor;
An explanation for the inter-species variability of the photoprotective non-photochemical chlorophyll fluorescence quenching in diatoms by Johann Lavaud; Bernard Lepetit (294-302).
Diatoms are a major group of microalgae whose photosynthetic productivity supports a substantial part of the aquatic primary production. In their natural environment they have to cope with strong fluctuations of the light climate which can be harmful for photosynthesis. In order to prevent the damage of their photosynthetic machinery, diatoms use fast regulatory processes among which the non-photochemical quenching of chlorophyll a fluorescence (NPQ) is one of the most important. In a previous work, we highlighted differences in the kinetics and extent of NPQ between diatom species/strains originating from different aquatic habitats. We proposed that the NPQ differences observed between strains/species could potentially participate to their ecophysiological adaptation to the light environment of their respective natural habitat. In order to better understand the molecular bases of such differences, we compared the NPQ features of four strains/species of diatoms known for their NPQ discrepancy. We could identify new spectroscopic fingerprints concomitant to NPQ and the related xanthophyll cycle. These fingerprints helped us propose a molecular explanation for the NPQ differences observed between the diatom species/strains examined. The present work further strengthens the potential role of NPQ in the ecophysiology of diatoms.► Diatom inter-species differences in non-photochemical quenching (NPQ) were assessed. ► New spectroscopic fingerprints (Δ522 nm, Chl a 711) related to NPQ were found. ► They were successfully used for characterizing the NPQ inter-species differences. ► They demonstrate an additional regulatory feature of the NPQ in diatoms. ► This study strengthens the potential role of NPQ in the ecophysiology of diatoms.
Keywords: Diatom; Diatoxanthin; Ecophysiology; Photoprotection; Non-photochemical fluorescence quenching; Xanthophyll cycle;
Identification of several sub-populations in the pool of light harvesting proteins in the pennate diatom Phaeodactylum tricornutum by Kathi Gundermann; Matthias Schmidt; Wolfram Weisheit; Maria Mittag; Claudia Büchel (303-310).
Diatoms are major contributors to the photosynthetic productivity of marine phytoplankton. In these organisms, fucoxanthin-chlorophyll proteins (FCPs) serve as light-harvesting proteins. We have explored the FCP complexes in Phaeodactylum tricornutum under low light (LL) and high light (HL) conditions. Sub-fractionating the pool of major FCPs yielded different populations of trimeric complexes. Only Lhcf and Lhc-like proteins were found in the trimers. Under LL, the first polypeptide fraction contained six different Lhcfs and was mainly composed of Lhcf10. It was characterised by the highest amount of fucoxanthin (Fx). The second was dominated by Lhcf10, Lhcf5 and Lhcf2, and had a lower Fx/Chl c ratio. Little Fx/Chl c also characterised the most abundant FCP complexes, found in fraction 3, composed mainly of Lhcf5. These FCPs bound Fx molecules with the strongest bathochromic shift. The last two fractions contained FCP complexes that were built mainly of Lhcf4, harbouring more Fx molecules that absorbed at shorter wavelengths. Under HL, the same main polypeptides were retrieved in the different fractions and spectroscopic features were almost identical except for a higher diadinoxanthin content. The total amount of Lhcf5 was reduced under HL, whereas the amount of the last two fractions and thereby Lhcf4 was increased. Lhcf11 was identified in different LL fractions, but not detected in any HL fraction, while two new Lhc-like proteins were only found under HL. This is the first report on different trimeric FCP complexes in pennate diatoms, which differ in polypeptide composition and pigmentation, and are differentially expressed by light.► Different trimeric light harvesting complexes exist in pennate diatoms. ► The trimers have different polypeptides and trimers of Lhcf5 are most abundant. ► The trimers differ in pigments: Lhcf5 binds less, Lhcf2/10 more fucoxanthin. ► Binding of fucoxanthin to Lhcf5 induces the most extreme bathochromic shift. ► The ratio between trimers differs in high light as compared to low light conditions.
Keywords: Diatom; Fucoxanthin chlorophyll protein; Light harvesting; Mass spectrometry; Photosynthesis;
Allophycocyanin and phycocyanin crystal structures reveal facets of phycobilisome assembly by Ailie Marx; Noam Adir (311-318).
X-ray crystal structures of the isolated phycobiliprotein components of the phycobilisome have provided high resolution details to the description of this light harvesting complex at different levels of complexity and detail. The linker-independent assembly of trimers into hexamers in crystal lattices of previously determined structures has been observed in almost all of the phycocyanin (PC) and allophycocyanin (APC) structures available in the Protein Data Bank. In this paper we describe the X-ray crystal structures of PC and APC from Synechococcus elongatus sp. PCC 7942, PC from Synechocystis sp. PCC 6803 and PC from Thermosynechococcus vulcanus crystallized in the presence of urea. All five structures are highly similar to other PC and APC structures on the levels of subunits, monomers and trimers. The Synechococcus APC forms a unique loose hexamer that may show the structural requirements for core assembly and rod attachment. While the Synechococcus PC assembles into the canonical hexamer, it does not further assemble into rods. Unlike most PC structures, the Synechocystis PC fails to form hexamers. Addition of low concentrations of urea to T. vulcanus PC inhibits this proteins propensity to form hexamers, resulting in a crystal lattice composed of trimers. The molecular source of these differences in assembly and their relevance to the phycobilisome structure is discussed.Display Omitted► Assembly of the phycobilisome is still poorely understood. ► Minor structural variances appear to facilitate hexamer formation. ► Five new phycocyanin and allophycocyanin crystal structures are presented. ► The structures reveal new attributes that lead to or inhibit hexamer formation. ► The small core linker protein modifies the allophycocyanin hexamer assembly.
Keywords: Photosynthesis; Cyanobacterium; X-ray crystallography; Complex assembly; Urea;
True wild type and recombinant wild type cytochrome c oxidase from Paracoccus denitrificans show a 20-fold difference in their catalase activity by Florian Hilbers; Iris von der Hocht; Bernd Ludwig; Hartmut Michel (319-327).
The four subunit (SU) aa 3 cytochrome c oxidase (CcO) from Paracoccus denitrificans is one of the terminal enzymes of the respiratory chain. Its binuclear active center, residing in SU I, contains heme a 3 and CuB. Apart from its oxygen reductase activity, the protein possesses a peroxidase and a catalase activity. To compare variants and the wild type (WT) protein in a more stringent way, a recombinant (rec.) WT strain was constructed, carrying the gene for SU I on a low copy number plasmid. This rec. WT showed no difference in oxygen reductase activity compared to the American Type Culture Collection (ATCC) WT CcO but surprisingly its catalase activity was increased by a factor of 20. The potential over-production of SU I might impair the correct insertion of heme a 3 and CuB because of a deficiency in metal inserting chaperones. An altered distance between heme a 3 and CuB and variations in protein structure are possible reasons for the observed increased catalase activity. The availability of chaperones was improved by cloning the genes ctaG and surf1c on the same plasmid as the SU I gene. The new rec. WT CcO showed in fact a reduced catalase activity. Using differential scanning calorimetry no significant difference in thermal stability between the ATCC WT CcO and the rec. WT CcO was detected. However, upon aging the thermal stability of the rec. WT CcO was reduced compared to that of the ATCC WT CcO pointing to a decreased structural stability of the rec. WT CcO.► Recombinant cytochrome c oxidase produced homologously was compared to the wild type. ► The recombinant protein shows a 20-fold increase in the catalase side reaction. ► Adjusting the chaperone levels leads to a significant decrease of the catalase activity. ► Well adjusted chaperone levels are required to achieve wild type activities.
Keywords: Bioenergetics; Hydrogen Peroxide; Cytochrome c Oxidase; Paracoccus denitrificans;
Effects of dehydration on light-induced conformational changes in bacterial photosynthetic reaction centers probed by optical and differential FTIR spectroscopy by Marco Malferrari; Alberto Mezzetti; Francesco Francia; Giovanni Venturoli (328-339).
Following light-induced electron transfer between the primary donor (P) and quinone acceptor (QA) the bacterial photosynthetic reaction center (RC) undergoes conformational relaxations which stabilize the primary charge separated state P+QA −. Dehydration of RCs from Rhodobacter sphaeroides hinders these conformational dynamics, leading to acceleration of P+QA − recombination kinetics [Malferrari et al., J. Phys. Chem. B 115 (2011) 14732-14750]. To clarify the structural basis of the conformational relaxations and the involvement of bound water molecules, we analyzed light-induced P+QA −/PQA difference FTIR spectra of RC films at two hydration levels (relative humidity r = 76% and r = 11%). Dehydration reduced the amplitude of bands in the 3700–3550 cm− 1 region, attributed to water molecules hydrogen bonded to the RC, previously proposed to stabilize the charge separation by dielectric screening [Iwata et al., Biochemistry 48 (2009) 1220–1229]. Other features of the FTIR difference spectrum were affected by partial depletion of the hydration shell (r = 11%), including contributions from modes of P (9-keto groups), and from NH or OH stretching modes of amino acidic residues, absorbing in the 3550–3150 cm− 1 range, a region so far not examined in detail for bacterial RCs. To probe in parallel the effects of dehydration on the RC conformational relaxations, we analyzed by optical absorption spectroscopy the kinetics of P+QA − recombination following the same photoexcitation used in FTIR measurements (20 s continuous illumination). The results suggest a correlation between the observed FTIR spectral changes and the conformational rearrangements which, in the hydrated system, strongly stabilize the P+QA − charge separated state over the second time scale.► P+QA −/PQA IR spectra of reaction centers were analyzed in the 4000–1200 cm− 1 region. ► The effect of dehydration has been studied by controlling the relative humidity. ► Dehydration decreases difference bands attributed to bound water molecules. ► Dehydration affects vibrational modes of P and NH or OH groups of the protein. ► Spectral changes correlate with the stability of the light-induced P+QA − state.
Keywords: Difference FTIR spectroscopy; Protein hydration; Bacterial reaction center; Conformational dynamics; Charge recombination; Dielectric relaxation;
Balancing photosynthetic electron flow is critical for cyanobacterial acclimation to nitrogen limitation by Eitan Salomon; Leeat Bar-Eyal; Shir Sharon; Nir Keren (340-347).
Nitrogen limitation forces photosynthetic organisms to reallocate available nitrogen to essential functions. At the same time, it increases the probability of photo-damage by limiting the rate of energy-demanding metabolic processes, downstream of the photosynthetic apparatus. Non-diazotrophic cyanobacteria cope with this situation by decreasing the size of their phycobilisome antenna and by modifying their photosynthetic apparatus. These changes can serve two purposes: to provide extra amino-acids and to decrease excitation pressure. We examined the effects of nitrogen limitation on the form and function of the photosynthetic apparatus. Our aim was to study which of the two demands serve as the driving force for the remodeling of the photosynthetic apparatus, under different growth conditions. We found that a drastic reduction in light intensity allowed cells to maintain a more functional photosynthetic apparatus: the phycobilisome antenna was bigger, the activity of both photosystems was higher and the levels of photosystem (PS) proteins were higher. Pre-acclimating cells to Mn limitation, under which the activity of both PSI and PSII is diminished, results in a very similar response. The rate of PSII photoinhibition, in nitrogen limited cells, was found to be directly related to the activity of the photosynthetic apparatus. These data indicate that, under our experimental conditions, photo-damage avoidance was the more prominent determinant during the acclimation process. The combinations of limiting factors tested here is by no means artificial. Similar scenarios can take place under environmental conditions and should be taken into account when estimating nutrient limitations in nature.► Cyanobacteria acclimate to -N conditions by decreasing antenna and RC content. ► Decreasing excitation energy pressure resulted in less photodamage under -N conditions. ► Photodamage avoidance is important to survive in N limited environments.
Keywords: Cyanobacteria; Nitrogen limitation; Mn limitation; Photosynthesis; Photoinhibition;
Characterization of the Synechocystis PCC 6803 Fluorescence Recovery Protein involved in photoprotection by Michal Gwizdala; Adjélé Wilson; Amin Omairi-Nasser; Diana Kirilovsky (348-354).
Under high irradiance, most cyanobacteria induce a photoprotective mechanism that decreases the energy arriving at the photosynthetic reaction centers to avoid the formation of dangerous species of oxygen. This mechanism which rapidly increases the heat dissipation of excess energy at the level of the cyanobacterial antenna, the phycobilisomes, is triggered by the photoactivation of the Orange Carotenoid Protein (OCP). Under low light conditions, the Fluorescence Recovery Protein (FRP) mediates the recovery of the full antenna capacity by accelerating the deactivation of the OCP. Several FRP Synechocystis mutants were constructed and characterized in terms of the OCP-related photoprotective mechanism. Our results demonstrate that Synechocystis FRP starts at Met26 and not at Met1 (according to notation in Cyanobase) as was previously suggested. Moreover, changes in the genomic region upstream the ATG encoding for Met26 influenced the concentration of OCP in cells. A long FRP (beginning at Met1) is synthesized in Synechocystis cells when the frp gene is under the control of the psbA2 promoter but it is less active than the shorter protein. Overexpression of the short frp gene in Synechocystis enabled short FRP isolation from the soluble fraction. However, the high concentration of FRP in this mutant inhibited the induction of the photoprotective mechanism by decreasing the concentration of the activated OCP. Therefore, the amplitude of photoprotection depends on not only OCP concentration but also on that of FRP. The synthesis of FRP and OCP must be strictly regulated to maintain a low FRP to OCP ratio to allow efficient photoprotection.► The Fluorescence Recovery Protein (FRP) switches off OCP-related photoprotection. ► It was proposed that Synechocystis FRP has a 25 amino-acid N-terminal prolongation. ► Our work shows that FRP has no such prolongation and starts at Met26. ► Short FRP-starting at Met26 can be isolated from Synechocystis and is active. ► High FRP to OCP ratio inhibits photoprotection.
Keywords: Cyanobacteria; Orange Carotenoid Protein; Non-photochemical quenching; Photoprotection; Photosynthesis; Phycobilisome;
The negative feedback molecular mechanism which regulates excitation level in the plant photosynthetic complex LHCII: Towards identification of the energy dissipative state by Monika Zubik; Rafal Luchowski; Michal Puzio; Ewa Janik; Joanna Bednarska; Wojciech Grudzinski; Wieslaw I. Gruszecki (355-364).
Overexcitation of the photosynthetic apparatus is potentially dangerous because it can cause oxidative damage. Photoprotection realized via the feedback de-excitation in the pigment–protein light-harvesting complex LHCII, embedded in the chloroplast lipid environment, was studied with use of the steady-state and time-resolved fluorescence spectroscopy techniques. Illumination of LHCII results in the pronounced singlet excitation quenching, demonstrated by decreased quantum yield of the chlorophyll a fluorescence and shortening of the fluorescence lifetimes. Analysis of the 77 K chlorophyll a fluorescence emission spectra reveals that the light-driven excitation quenching in LHCII is associated with the intensity increase of the spectral band in the region of 700 nm, relative to the principal band at 680 nm. The average chlorophyll a fluorescence lifetime at 700 nm changes drastically upon temperature decrease: from 1.04 ns at 300 K to 3.63 ns at 77 K. The results of the experiments lead us to conclude that: (i) the 700 nm band is associated with the inter-trimer interactions which result in the formation of the chlorophyll low-energy states acting as energy traps and non-radiative dissipation centers; (ii) the Arrhenius analysis, supported by the results of the FTIR measurements, suggests that the photo-reaction can be associated with breaking of hydrogen bonds. Possible involvement of photo-isomerization of neoxanthin, reported previously (Biochim. Biophys. Acta 1807 (2011) 1237-1243) in generation of the low-energy traps in LHCII is discussed.Display Omitted► Illumination of LHCII induces excitation quenching. ► Excitation quenching is associated with appearance of the emission band at 700 nm. ► The 700 nm band is attributed to inter-trimer LHCII interactions.
Keywords: Photosynthesis; Photoprotection; Xanthophyll; Fluorescence quenching; LHCII;
Role of the -PEWY-glutamate in catalysis at the Qo-site of the Cyt bc 1 complex by Doreen Victoria; Rodney Burton; Antony R. Crofts (365-386).
We re-examine the pH dependence of partial processes of ubihydroquinone (QH2) turnover in Glu-295 mutants in Rhodobacter sphaeroides to clarify the mechanistic role. In more crippled mutants, the bell-shaped pH profile of wildtype was replaced by dependence on a single pK at ~ 8.5 favoring electron transfer. Loss of the pK at 6.5 reflects a change in the rate-limiting step from the first to the second electron transfer. Over the range of pH 6–8, no major pH dependence of formation of the initial reaction complex was seen, and the rates of bypass reactions were similar to the wildtype. Occupancy of the Qo-site by semiquinone (SQ) was similar in the wildtype and the Glu → Trp mutant. Since heme b L is initially oxidized in the latter, the bifurcated reaction can still occur, allowing estimation of an empirical rate constant < 103 s− 1 for reduction of heme b L by SQ from the domain distal from heme b L, a value 1000-fold smaller than that expected from distance. If the pK ~ 8.5 in mutant strains is due to deprotonation of the neutral semiquinone, with Q•− as electron donor to heme b L, then in wildtype this low value would preclude mechanisms for normal flux in which semiquinone is constrained to this domain. A kinetic model in which Glu-295 catalyzes H+ transfer from QH•, and delivery of the H+ to exit channel(s) by rotational displacement, and facilitates rapid electron transfer from SQ to heme b L by allowing Q•− to move closer to the heme, accounts well for the observations.► In mutants at the -PEWY- glutamate, the second electron transfer becomes limiting. ► With k cat ~ 103 s− 1, electron transfer from the distal domain is kinetically incompetent. ► Bifurcated rate at the Qo-site is pH independent below 8, and increases with pK ~ 8.5. ► There is no marked pH-dependence of ES formation over the range < 8 in mutants. ► E295 catalyzes H+ exit and likely acts as a gate for semiquinone migration in the site.
Keywords: Bifurcated reaction of Q-cycle; Control and gating; Semiquinone occupancy; H+ exit pathway; Kinetic model;
Formation of an unusually short hydrogen bond in photoactive yellow protein by Keisuke Saito; Hiroshi Ishikita (387-394).
The photoactive chromophore of photoactive yellow protein (PYP) is p-coumaric acid (pCA). In the ground state, the pCA chromophore exists as a phenolate anion, which is H-bonded by protonated Glu46 (OGlu46–O pCA = ~ 2.6 Å) and protonated Tyr42. On the other hand, the OGlu46–O pCA H-bond was unusually short (OGlu46–O pCA = 2.47 Å) in the intermediate pRCW state observed in time-resolved Laue diffraction studies. To understand how the existence of the unusually short H-bond is energetically possible, we analyzed the H-bond energetics adopting a quantum mechanical/molecular mechanical (QM/MM) approach based on the atomic coordinates of the PYP crystal structures. In QM/MM calculations, the OGlu46–O pCA bond is 2.60 Å in the ground state, where Tyr42 donates an H-bond to pCA. In contrast, when the hydroxyl group of Tyr42 is flipped away from pCA, the H-bond was significantly shortened to 2.49 Å in the ground state. The same H-bond pattern reproduced the unusually short H-bond in the pRCW structure (OGlu46–O pCA = 2.49 Å). Intriguingly, the potential-energy profile resembles that of a single-well H-bond, suggesting that the pK a values of the donor (Glu46) and acceptor (pCA) moieties are nearly equal. The present results indicate that the “equal pK a” requirement for formation of single-well or low-barrier H-bond (LBHB) is satisfied only when Tyr42 does not donate an H-bond to pCA, and argue against the possibility that the OGlu46–O pCA bond is an LBHB in the ground state, where Tyr42 donates an H-bond to pCA.► The short OGlu46–O pCA in pRCW was reproduced when the OTyr42–O pCA H-bond is absent. ► The energy profile of OGlu46–O pCA in pRCW resembles that of a single-well H bond. ► The barrierless OGlu46–O pCA H-bond in pRCW facilitates proton transfer. ► OGlu46–O pCA is a standard H-bond when Tyr42 forms an H-bond with pCA in pG.
Keywords: Low-barrier hydrogen bond; Proton transfer; Photoactive yellow protein; Laue diffraction crystallography; 1H NMR;
Does acetogenesis really require especially low reduction potential? by Arren Bar-Even (395-400).
Acetogenesis is one of the oldest metabolic processes on Earth, and still has a major global significance. In this process, acetate is produced via the reduction and condensation of two carbon dioxide molecules. It has long been assumed that acetogenesis requires ferredoxin with an exceptionally low reduction potential of ≈ − 500 mV in order to drive CO2 reduction to CO and the reductive carboxylation of acetyl-CoA to pyruvate. However, no other metabolic pathway requires electron donors with such low reduction potential. Is acetogenesis a special case, necessitating unique cellular conditions? In this paper, I suggest that it is not. Rather, by keeping CO as a bound metabolite, the CO-dehydrogenase-acetyl-CoA-synthase complex can couple the unfavorable CO2 reduction to CO with the favorable acetyl-CoA synthesis, thus enabling the former process to proceed using ferredoxin of moderate reduction potential of − 400 mV. I further show that pyruvate synthesis can also take place using the same ferredoxins. In fact, the synthesis of pyruvate from CO2, methylated-protein-carrier and − 400 mV ferredoxins is an energy-neutral process. These findings suggest that acetogenesis can take place at normal cellular redox state. Mechanistic coupling of reactions as suggested here can flatten energetic landscapes and diminish thermodynamic barriers and can be another role for enzymatic complexes common in nature and a useful tool for metabolic engineering.► Acetogenesis is assumed to operate under very low reduction potential ► CO-dehydrogenase-acetyl-CoA-synthase complex diminishes internal energetic barriers ► Acetogenesis can therefore operate under normal reduction potential ► Acetogenesis and pyruvate synthesis are reversible at cellular conditions ► Enzymatic complexes present a general mechanism to lower energetic barriers
Keywords: Wood–Ljungdahl pathway; CO-dehydrogenase-acetyl-CoA-synthase complex; Ferredoxin; Enzymatic complex; Reduction potential; Thermodynamics;
The function and the role of the mitochondrial glycerol-3-phosphate dehydrogenase in mammalian tissues by Tomáš Mráček; Zdeněk Drahota; Josef Houštěk (401-410).
Mitochondrial glycerol-3-phosphate dehydrogenase (mGPDH) is not included in the traditional textbook schemes of the respiratory chain, reflecting the fact that it is a non-standard, tissue-specific component of mammalian mitochondria. But despite its very simple structure, mGPDH is a very important enzyme of intermediary metabolism and as a component of glycerophosphate shuttle it functions at the crossroads of glycolysis, oxidative phosphorylation and fatty acid metabolism. In this review we summarize the present knowledge on the structure and regulation of mGPDH and discuss its metabolic functions, reactive oxygen species production and tissue and organ specific roles in mammalian mitochondria at physiological and pathological conditions.► With 74 kDa mGPDH is the most simple component of mammalian respiratory chain. ► Highly tissue specific mGPDH forms glycerophosphate shuttle with cytosolic cGPDH. ► mGPDH connects glycolysis, fatty acid metabolism and oxidative phosphorylation. ► mGPDH is prone to electron leak and represents important site of ROS generation. ► Pathophysiological aspects of organ specific roles of mGPDH are discussed.
Keywords: GPD2 gene; Mitochondrial glycerol-3-phosphate dehydrogenase; Glycerophosphate shuttle; ROS production; Pathophysiology;
High-light vs. low-light: Effect of light acclimation on photosystem II composition and organization in Arabidopsis thaliana by Roman Kouřil; Emilie Wientjes; Jelle B. Bultema; Roberta Croce; Egbert J. Boekema (411-419).
The structural response of photosystem II (PSII) and its light-harvesting proteins (LHCII) in Arabidopis thaliana after long-term acclimation to either high or low light intensity was characterized. Biochemical and structural analysis of isolated thylakoid membranes by electron microscopy indicates a distinctly different response at the level of PSII and LHCII upon plant acclimation. In high light acclimated plants, the C2S2M2 supercomplex, which is the dominating form of PSII in Arabidopsis, is a major target of structural re-arrangement due to the down-regulation of Lhcb3 and Lhcb6 antenna proteins. The PSII ability to form semi-crystalline arrays in the grana membrane is strongly reduced compared to plants grown under optimal light conditions. This is due to the structural heterogeneity of PSII supercomplexes rather than to the action of PsbS protein as its level was unexpectedly reduced in high light acclimated plants. In low light acclimated plants, the architecture of the C2S2M2 supercomplex and its ability to form semi-crystalline arrays remained unaffected but the density of PSII in grana membranes is reduced due to the synthesis of additional LHCII proteins. However, the C2S2M2 supercomplexes in semi-crystalline arrays are more densely packed, which can be important for efficient energy transfer between PSII under light limiting conditions.Display Omitted► Acclimation to high light intensity leads to structural re-arrangements of PSII. ► Formation of PSII semi-crystalline arrays is reduced in high light acclimated plants. ► Amount of PsbS protein is reduced in high light acclimated plants. ► PSII in semi-crystalline arrays is densely packed in low light acclimated plants.
Keywords: Photosystem II supercomplex; Thylakoid membrane; Electron microscopy; Light acclimation;
LHCII is an antenna of both photosystems after long-term acclimation by Emilie Wientjes; Herbert van Amerongen; Roberta Croce (420-426).
LHCII, the most abundant membrane protein on earth, is the major light-harvesting complex of plants. It is generally accepted that LHCII is associated with Photosystem II and only as a short-term response to overexcitation of PSII a subset moves to Photosystem I, triggered by its phosphorylation (state1 to state2 transition). However, here we show that in most natural light conditions LHCII serves as an antenna of both Photosystem I and Photosystem II and it is quantitatively demonstrated that this is required to achieve excitation balance between the two photosystems. This allows for acclimation to different light intensities simply by regulating the expression of LHCII genes only. It is demonstrated that indeed the amount of LHCII that is bound to both photosystems decreases when growth light intensity increases and vice versa. Finally, time-resolved fluorescence measurements on the photosynthetic thylakoid membranes show that LHCII is even a more efficient light harvester when associated with Photosystem I than with Photosystem II.► LHCII is associated with Photosystem I in nearly all light conditions. ► Antenna size of PSI and PSII is regulated in parallel by LHCII during acclimation. ► LHCII transfers excitation energy faster to PSI than to PSII.
Keywords: Excitation-energy transfer; Light-harvesting complex; Photosystem; State transitions; Supercomplex;
Antagonist effect between violaxanthin and de-epoxidated pigments in nonphotochemical quenching induction in the qE deficient brown alga Macrocystis pyrifera by Héctor Ocampo-Alvarez; Ernesto García-Mendoza; Govindjee (427-437).
Nonphotochemical quenching (NPQ) of Photosystem II fluorescence is one of the most important photoprotection responses of phototropic organisms. NPQ in Macrocystis pyrifera is unique since the fast induction of this response, the energy dependent quenching (qE), is not present in this alga. In contrast to higher plants, NPQ in this organism is much more strongly related to xanthophyll cycle (XC) pigment interconversion. Characterization of how NPQ is controlled when qE is not present is important as this might represent an ancient response to light stress. Here, we describe the influence of the XC pigment pool (ΣXC) size on NPQ induction in M. pyrifera. The sum of violaxanthin (Vx) plus antheraxanthin and zeaxanthin (Zx) represents the ΣXC. This pool was three-fold larger in blades collected at the surface of the water column (19 mol mol− 1 Chl a × 100) than in blades collected at 6 m depth. Maximum NPQ was not different in samples with a ΣXC higher than 12 mol mol− 1 Chl a × 100; however, NPQ induction was faster in blades with a large ΣXC. The increase in the NPQ induction rate was associated with a faster Vx to Zx conversion. Further, we found that NPQ depends on the de-epoxidation state of the ΣXC, not on the absolute concentration of Zx and antheraxanthin. Thus, there was an antagonist effect between Vx and de-epoxidated xanthophylls for NPQ. These results indicate that in the absence of qE, a large ΣXC is needed in M. pyrifera to respond faster to light stress conditions.► A model of nonphotochemical (NPQ) quenching control in M. pyrifera is proposed. ► NPQ in the qE deficient M. pyrifera is related to the de-epoxidation state ratio. ► Violaxanthin and de-epoxidated xanthophylls compete for the quenching sites. ► Large xanthophyll cycle pigment pool accelerates the de-epoxidation of violaxanthin. ► High light acclimated blades have a large xanthophyll cycle pigment pool.
Keywords: Xanthophyll cycle pool size; Zeaxanthin-dependent quenching (qZ); De-epoxidation rate control; Brown alga; Macrocystis pyrifera;
Electronic structure of S2 state of the oxygen-evolving complex of photosystem II studied by PELDOR by Mizue Asada; Hiroki Nagashima; Faisal Hammad Mekky Koua; Jian-Ren Shen; Asako Kawamori; Hiroyuki Mino (438-445).
Photosynthetic water splitting is catalyzed by a Mn4CaO5 cluster in photosystem II, whose structure was recently determined at a resolution of 1.9 Å [Umena, Y. et al. 2011, Nature, 473:55–60]. To determine the electronic structure of the Mn4CaO5 cluster, pulsed electron–electron double resonance (PELDOR) measurements were performed for the tyrosine residue YD • and S2 state signals with non-oriented and oriented photosystem II (PS II) samples. Based on these measurements, the spin density distributions were calculated by comparing with the experimental results. The best fitting parameters were obtained with a model in which Mn1 has a large positive projection, Mn3 has a small positive projection, and Mn2 and Mn4 have negative projections (the numbering of Mni (i = 1–4) is based on the crystal structure at a 1.9 Å resolution), which yielded spin projections of 1.97, − 1.20, 1.19 and − 0.96 for Mn1–4 ions. The results show that the Mn1 ion, which is coordinated by H332, D342 and E189, has a valence of Mn(III) in the S2 state. The sign of the exchange interactions J 13 is positive, and the other signs are negative.Display Omitted► We measured PELDOR between tyrosine and Mn cluster in S2 state of photosystem II. ► Angular dependence of PELDOR signals was detected in oriented membranes. ► Spin projections of Mn ions were directly estimated from the 1.9 Å crystal structure. ► The Mn ion coordinated by H332, D342 and D189 was assigned to be Mn(III). ► Signs of magnetic couplings between the Mn ions were clarified.
Keywords: Photosystem II; Oxygen-evolving complex; Mn cluster; EPR; PELDOR;
Partitioning of superoxide and hydrogen peroxide production by mitochondrial respiratory complex I by Vera G. Grivennikova; Andrei D. Vinogradov (446-454).
Membrane-bound respiratory complex I in inside-out submitochondrial particles (SMP) catalyzes both superoxide and hydrogen peroxide formation in NADH- and/or succinate-supported reactions. At optimal NADH concentration (50 μM), the complex I-mediated process results in a formation of two superoxide anions and H2O2 as the reaction products in approximately 0.7 ratio. Almost the same ratio is found for purified complex I (0.6) and for the aerobic succinate-supported reverse electron transfer reaction. Superoxide production is depressed at high, more physiologically relevant NADH concentrations, whereas hydrogen peroxide formation is insensitive to the elevated level of NADH. The rates of H2O2 formation at variable NAD+/NADH ratios satisfactorily fit the Nernst equation for a single reactive two-electron donor component equilibrated with ambient midpoint redox potential of − 347 mV (0.13 NAD+/NADH ratio, pH 8.0). Half-maximal superoxide production rate proceeds at significantly higher NAD+/NADH ratio (0.33). Guanidine strongly stimulates NADH-supported hydrogen peroxide and superoxide production at any NADH concentration and activates NADH:ferricyanide and inhibits NADH:hexaammineruthenium (III) reductase activities while showing no effects on NADH oxidase of SMP. In the low range of NADH concentration, superoxide production rate shows a simple hyperbolic dependence on NADH with apparent K m NADH of 0.5 μM, whereas sigmoidal dependence of hydrogen peroxide production is seen with half-maximal rate at 25 μM NADH. We interpret the data as to suggest that at least two sites participate in complex I-mediated ROS generation: FMNH– that produces hydrogen peroxide, and an iron–sulfur center (likely N-2) that produces superoxide anion.► Mammalian complex I generates both superoxide and hydrogen peroxide. ► Relative contribution of either species depends on NADH concentration. ► Guanidine stimulates complex I-mediated ROS production.
Keywords: Respiratory complex I; Dihydrolipoamide dehydrogenase; Hydrogen peroxide; Superoxide anion; Pyridine nucleotides; Guanidine;
Iron/sulfur proteins biogenesis in prokaryotes: Formation, regulation and diversity by Béatrice Roche; Laurent Aussel; Benjamin Ezraty; Pierre Mandin; Béatrice Py; Frédéric Barras (455-469).
Iron/sulfur centers are key cofactors of proteins intervening in multiple conserved cellular processes, such as gene expression, DNA repair, RNA modification, central metabolism and respiration. Mechanisms allowing Fe/S centers to be assembled, and inserted into polypeptides have attracted much attention in the last decade, both in eukaryotes and prokaryotes. Basic principles and recent advances in our understanding of the prokaryotic Fe/S biogenesis ISC and SUF systems are reviewed in the present communication. Most studies covered stem from investigations in Escherichia coli and Azotobacter vinelandii. Remarkable insights were brought about by complementary structural, spectroscopic, biochemical and genetic studies. Highlights of the recent years include scaffold mediated assembly of Fe/S cluster, A-type carriers mediated delivery of clusters and regulatory control of Fe/S homeostasis via a set of interconnected genetic regulatory circuits. Also, the importance of Fe/S biosynthesis systems in mediating soft metal toxicity was documented. A brief account of the Fe/S biosynthesis systems diversity as present in current databases is given here. Moreover, Fe/S biosynthesis factors have themselves been the object of molecular tailoring during evolution and some examples are discussed here. An effort was made to provide, based on the E. coli system, a general classification associating a given domain with a given function such as to help next search and annotation of genomes. This article is part of a Special Issue entitled: Metals in Bioenergetics and Biomimetics Systems.► Fe/S assembly understanding was enhanced by structural analysis of IscU–IscS complex. ► A-type carriers allow for the delivery of preformed clusters. ► Fe/S homeostasis is controlled by IscR, Fur and RyhB. ► Metal toxicity is mediated via Fe/S proteins. ► Fe/S biosynthesis systems show diversity in both genetic organization and factors content.
Keywords: Fe―S cluster biosynthesis; Fe―S cluster homeostasis; Fe―S regulation; Fe―S domains; Fe―S cluster and pathogens; Metal toxicity;