BBA - Bioenergetics (v.1777, #1)

Oxygen sensors in context by Jeremy P.T. Ward (1-14).
The ability to adapt to changes in the availability of O2 provides a critical advantage to all O2-dependent lifeforms. In mammals it allows optimal matching of the O2 requirements of the cells to ventilation and O2 delivery, underpins vital changes to the circulation during the transition from fetal to independent, air-breathing life, and provides a means by which dysfunction can be limited or prevented in disease. Certain tissues such as the carotid body, pulmonary circulation, neuroepithelial bodies and fetal adrenomedullary chromaffin cells are specialised for O2 sensing, though most others show for example alterations in transcription of specific genes during hypoxia. A number of mechanisms are known to respond to variations in PO2 over the physiological range, and have been proposed to fulfil the function as O2 sensors; these include modulation of mitochondrial oxidative phosphorylation and a number of O2-dependent synthetic and degradation pathways. There is however much debate as to their relative importance within and between specific tissues, whether their O2 sensitivity is actually appropriate to account for their proposed actions, and in particular their modus operandi. This review discusses our current understanding of how these mechanisms may operate, and attempts to put them into the context of the actual PO2 to which they are likely to be exposed. An important point raised is that the overall O2 sensitivity (P50) of any O2-dependent mechanism does not necessarily correlate with that of its O2 sensor, as the coupling function between the two may be complex and non-linear. In addition, although the bulk of the evidence suggests that mitochondria act as the key O2 sensor in carotid body, pulmonary artery and chromaffin cells, the signalling mechanisms by which alterations in their function are translated into a response appear to differ fundamentally, making a global unified theory of O2 sensing unlikely.
Keywords: Oxygen sensor; Mitochondria; Carotid body; Pulmonary circulation; Oxygen distribution; Hypoxic pulmonary vasoconstriction; Electron transport chain; Reactive oxygen species; Redox state;

Physiological bond formation and bond breaking events between proteins and ligands and their immediate consequences are difficult to synchronize and study in general. However, diatomic ligands can be photodissociated from heme, and thus in heme proteins ligand release and rebinding dynamics and trajectories have been studied on timescales of the internal vibrations of the protein that drive many biochemical reactions, and longer. The rapidly expanding number of characterized heme proteins involved in a large variety of functions allows comparative dynamics–structure–function studies. In this review, an overview is given of recent progress in this field, and in particular on initial sensing processes in signaling proteins, and on ligand and electron transfer dynamics in oxidases and cytochromes.
Keywords: Heme proteins; Femtosecond spectroscopy; Molecular dynamics; Heme-based sensors; Oxidases; Cytochrome;

ATP synthesis without R210 of subunit a in the Escherichia coli ATP synthase by Robert R. Ishmukhametov; J. Blake Pond; Asma Al-Huqail; Mikhail A. Galkin; Steven B. Vik (32-38).
Interactions between subunit a and oligomeric subunit c are essential for the coupling of proton translocation to rotary motion in the ATP synthase. A pair of previously described mutants, R210Q/Q252R and P204T/R210Q/Q252R [L.P. Hatch, G.B. Cox and S.M. Howitt, The essential arginine residue at position 210 in the a subunit of the Escherichia coli ATP synthase can be transferred to position 252 with partial retention of activity, J. Biol. Chem. 270 (1995) 29407–29412] has been constructed and further analyzed. These mutants, in which the essential arginine of subunit a, R210, was switched with a conserved glutamine residue, Q252, are shown here to be capable of both ATP synthesis by oxidative phosphorylation, and ATP-driven proton translocation. In addition, lysine can replace the arginine at position 252 with partial retention of both activities. The pH dependence of ATP-driven proton translocation was determined after purification of mutant enzymes, and reconstitution into liposomes. Proton translocation by the lysine mutant, and to a lesser extent the arginine mutant, dropped off sharply above pH 7.5, consistent with the requirement for a positive charge during function. Finally, the rates of ATP synthesis and of ATP-driven proton translocation were completely inhibited by treatment with DCCD (N,N′-dicyclohexylcarbodiimide), while rates of ATP hydrolysis by the mutants were not significantly affected, indicating that DCCD modification disrupts the F1–Fo interface. The results suggest that minimal requirements for proton translocation by the ATP synthase include a positive charge in subunit a and a weak interface between subunit a and oligomeric subunit c.
Keywords: ATP synthase; F1Fo; Subunit a; Proton translocation; ATP synthesis; Rotary motor;

Mitochondrial biogenesis in fast skeletal muscle of CK deficient mice by A. Vaarmann; D. Fortin; V. Veksler; I. Momken; R. Ventura-Clapier; A. Garnier (39-47).
Creatine kinase (CK) is a phosphotransfer kinase that catalyzes the reversible transfer of a phosphate moiety between ADP and creatine and that is highly expressed in skeletal muscle. In fast glycolytic skeletal muscle, deletion of the cytosolic M isoform of CK in mice (M-CK−/−) leads to a massive increase in the oxidative capacity and of mitochondrial volume. This study was aimed at investigating the transcriptional pathways leading to mitochondrial biogenesis in response to CK deficiency. Wild type and M-CK−/− mice of eleven months of age were used for this study. Gastrocnemius muscles of M-CK−/− mice exhibited a dramatic increase in citrate synthase (+ 120%) and cytochrome oxidase (COX, + 250%) activity, and in mitochondrial DNA (+ 60%), showing a clear activation of mitochondrial biogenesis. Similarly, mRNA expression of the COXI (mitochondria-encoded) and COXIV (nuclear-encoded) subunits were increased by + 103 and + 94% respectively. This was accompanied by an increase in the expression of the nuclear respiratory factor (NRF2α) and the mitochondrial transcription factor (mtTFA). Expression of the co-activator PGC-1α, a master gene in mitochondrial biogenesis was not significantly increased while that of PGC-1β and PRC, two members of the same family, was moderately increased (+ 45% and + 55% respectively). While the expression of the modulatory calcineurin-interacting protein 1 (MCIP1) was dramatically decreased (− 68%) suggesting inactivation of the calcineurin pathway, the metabolic sensor AMPK was activated (+ 86%) in M-CK−/− mice. These results evidence that mitochondrial biogenesis in response to a metabolic challenge exhibits a unique pattern of regulation, involving activation of the AMPK pathway.
Keywords: Skeletal muscle; CK-KO mice; Energy metabolism; AMPK; Transcription;

Modified glutamine catabolism in macrophages of Ucp2 knock-out mice by Tobias Nübel; Yalin Emre; Daniel Rabier; Bernadette Chadefaux; Daniel Ricquier; Frédéric Bouillaud (48-54).
Uncoupling protein 2 (UCP2) belongs to a family of transporters of the mitochondrial inner membrane and is reported to uncouple respiration from ATP synthesis. Our observation that the amino acid glutamine specifically induces UCP2 protein expression prompted us to investigate metabolic consequences of a UCP2 knockdown (Ucp2-KO) when glutamine is offered as a substrate. We found that Ucp2-KO macrophages incubated in the presence of glutamine exhibit a lower ammonium release, a decreased respiratory rate, and an intracellular accumulation of aspartate. Therefore, we conclude that UCP2 expression is required for efficient oxidation of glutamine in macrophages. This role of UCP2 in glutamine metabolism appears independent from the uncoupling activity of UCP2.
Keywords: UCP2; Macrophage; Uncoupling protein; Mitochondria; Glutamine; Glutaminolysis;

Energy transfer processes in Gloeobacter violaceus PCC 7421 that possesses phycobilisomes with a unique morphology by Makio Yokono; Seiji Akimoto; Kohei Koyama; Tohru Tsuchiya; Mamoru Mimuro (55-65).
We examined energy transfer dynamics in phycobilisomes (PBSs) of cyanobacteria in relation to the morphology and pigment compositions of PBSs. We used Gloeobacter violaceus PCC 7421 and measured time-resolved fluorescence spectra in three types of samples, i.e., intact cells, PBSs, and rod assemblies separated from cores. Fremyella diplosiphon, a cyanobacterial species well known for its complementary chromatic adaptation, was used for comparison after growing under red or green light. Spectral data were analyzed by the fluorescence decay-associated spectra with components common in lifetimes with a time resolution of 3 ps/channel and a spectral resolution of 2 nm/channel. This ensured a higher resolution of the energy transfer kinetics than those obtained by global analysis with fewer sampling intervals. We resolved four spectral components in phycoerythrin (PE), three in phycocyanin (PC), two in allophycocyanin, and two in photosystem II. The bundle-like PBSs of G. violaceus showed multiple energy transfer pathways; fast (≈ 10 ps) and slow (≈ 100 ps and ≈ 500 ps) pathways were found in rods consisting of PE and PC. Energy transfer time from PE to PC was two times slower in G. violaceus than in F. diplosiphon grown under green light.
Keywords: Chromatic adaptation; Energy transfer; Phycobilisome; Time-resolved fluorescence; Gloeobacter violaceus;

Control of cell respiration by nitric oxide in Ataxia Telangiectasia lymphoblastoid cells by Alessandra Masci; Daniela Mastronicola; Marzia Arese; Maria Piane; Andrea De Amicis; Thomas J.J. Blanck; Luciana Chessa; Paolo Sarti (66-73).
Ataxia Telangiectasia (AT) patients are particularly sensitive to oxidative–nitrosative stress. Nitric oxide (NO) controls mitochondrial respiration via the reversible inhibition of complex IV. The mitochondrial response to NO of AT lymphoblastoid cells was investigated. Cells isolated from three patients and three intrafamilial healthy controls were selected showing within each group a normal diploid karyotype and homogeneous telomere length. Different complex IV NO-inhibition patterns were induced by varying the electron flux through the respiratory chain, using exogenous cell membrane permeable electron donors. Under conditions of high electron flux the mitochondrial NO inhibition of respiration was greater in AT than in control cells (P  ≤ 0.05). This property appears peculiar to AT, and correlates well to the higher concentration of cytochrome c detected in the AT cells. This finding is discussed on the basis of the proposed mechanism of reaction of NO with complex IV. It is suggested that the peculiar response of AT mitochondria to NO stress may be relevant to the mitochondrial metabolism of AT patients.
Keywords: Nitrosative stress; Bioenergetics; Cytochrome c oxidase; Genetic disease; Reaction mechanism; Respiratory chain;

In Thermosynechococcus elongatus BP-1, which is the preferred organism in recent structural studies of PSII, three psbA and two psbD genes code for three D1 and one D2 protein isoforms, respectively. The regulation and function of these genes and protein products is largely unknown. Therefore, we used quantitative RT-PCR to follow changes in the mRNA level of the respective genes, in combination with biophysical measurements to detect changes in the electron transport activity of Photosystem II under exposure to different visible and UV light, and temperature conditions. In cells which are acclimated to 40 μmol m− 2s− 1 growth light conditions at 40 °C the main populations of the psbA and psbD transcripts arise from the psbA1 and psbD1 genes, respectively. When the temperature is raised to 60 °C psbA1 becomes the single dominating psbA mRNA species. Upon exposure of the cells to 500 μmol m− 2s− 1 intensity visible light psbA3 replaces psbA1 as the dominating psbA mRNA species, and psbD2 increases at the expense of psbD1. UV-B radiation also increases the abundance of psbA3, and psbD2 at the expense of psbA1 and psbD1, respectively. From the different extent of total D1 protein loss in the absence and presence of lincomycin it was estimated that the PsbA3 protein isoform replaces PsbA1 in about 65% of PSII centers after 2 h of high light acclimation. Under the conditions of different psbA transcript distributions chlorophyll fluorescence and thermoluminescence measurements were applied to monitor charge recombination characteristics of the S2QA and S2QB states. We obtained faster decay of flash-induced chlorophyll fluorescence in the presence of DCMU, as well as lower peak temperature of the Q and B thermoluminescence bands when PsbA3 replaced PsbA1 as the main D1 protein isoform. The relevance of dynamic changes in the abundance of psbA and psbD transcript levels, as well as D1 protein isoforms in the acclimation of T. elongatus to changing environmental conditions is discussed.
Keywords: psbA gene; psbD gene; D1 protein; Photosystem II; Photo-oxidative stress;

The projection structures of complex I and the I + III2 supercomplex from the C4 plant Zea mays were determined by electron microscopy and single particle image analysis to a resolution of up to 11 Å. Maize complex I has a typical L-shape. Additionally, it has a large hydrophilic extra-domain attached to the centre of the membrane arm on its matrix-exposed side, which previously was described for Arabidopsis and which was reported to include carbonic anhydrase subunits. A comparison with the X-ray structure of homotrimeric γ-carbonic anhydrase from the archaebacterium Methanosarcina thermophila indicates that this domain is also composed of a trimer. Mass spectrometry analyses allowed to identify two different carbonic anhydrase isoforms, suggesting that the γ-carbonic anhydrase domain of maize complex I most likely is a heterotrimer. Statistical analysis indicates that the maize complex I structure is heterogeneous: a less-abundant “type II” particle has a 15 Å shorter membrane arm and an additional small protrusion on the intermembrane-side of the membrane arm if compared to the more abundant “type I” particle. The I + III2 supercomplex was found to be a rigid structure which did not break down into subcomplexes at the interface between the hydrophilic and the hydrophobic arms of complex I. The complex I moiety of the supercomplex appears to be only of “type I”. This would mean that the “type II” particles are not involved in the supercomplex formation and, hence, could have a different physiological role.
Keywords: Complex I; Cytochrome c reductase; Carbonic anhydrase; Supercomplex; Electron microscopy;

Optical spectroscopic properties of the covalently linked chromophores of biliproteins are profoundly influenced by the state of the protein. This has been used to monitor the urea-induced denaturation of C-phycocyanin (CPC) from Mastigocladus laminosus and its subunits. Under equilibrium conditions, absorption, fluorescence and circular dichroism of the chromophores were monitored, as well as the circular dichroism of the polypeptide. Treatment of CPC trimers (αβ)3 resulted first in monomerization (αβ), which was followed by a complex unfolding process of the protein. Loss of chromophore fluorescence is the next process at increasing urea concentrations; it indicates increased flexibility of the chromophore while maintaining the native, extended conformation, and a less compact but still native-like packing of the protein in the regions sampled by the chromophores. This was followed by relaxation of the chromophores from the energetically unfavorable extended to a cyclic-helical conformation, as reported by absorption and CD in the visible range, indicating local loss of protein structure. Only then is the protein secondary structure lost, as reported by the far-UV CD. Sequential processes were also seen in the subunits, where again the chromophore–protein interactions were reduced before the unfolding of the protein. It is concluded that the bilin chromophores are intrinsic probes suitable to differentiate among different processes involved in protein denaturation.
Keywords: Biliprotein; Protein folding; Protein mobility; Protein dissociation; Chromophore–protein interaction; Optical spectroscopy;

Synchronized changes in transcript levels of genes activating cold exposure-induced thermogenesis in brown adipose tissue of experimental animals by Masahiro Watanabe; Takenori Yamamoto; Rei Kakuhata; Naoto Okada; Kazuaki Kajimoto; Naoshi Yamazaki; Masatoshi Kataoka; Yoshinobu Baba; Toshiaki Tamaki; Yasuo Shinohara (104-112).
To identify genes whose expression in brown adipose tissue (BAT) is up- or down-regulated in cold-exposed rats, we performed microarray analysis of RNA samples prepared from the BAT of cold-exposed rats and of rats kept at room temperature. Previously reported elevations of transcript levels of uncoupling protein (UCP1), type II iodothyronine deiodinase (DIO2), and type III adenylate cyclase (AC3) in the BAT of cold-exposed rats over those in that of rats maintained at room temperature were confirmed. In addition to these changes, remarkable elevations of the transcript levels of several genes that seemed to be associated with the processes of cell-cycle regulation and DNA replication were detected in the BAT of cold-exposed rats, possibly reflecting the significant proliferation of brown adipocytes in response to cold exposure. Up-regulation of the gene encoding sarcomeric mitochondrial type creatine kinase in the BAT of cold-exposed rats was also detected by microarray analysis, but subsequent Northern analysis revealed that the expression of not only the sarcomeric mitochondrial type enzyme, but also that of 2 other subtypes, i.e., cytoplasmic brain type and cytoplasmic muscle type, was elevated in the BAT of cold-exposed rats. Microarray analysis also revealed a significant expression of myoglobin in BAT and its elevation in the BAT of cold-exposed rats, and this result was supported by calibrated Northern analysis. On the contrary, several genes such as regulator of G-protein signaling 2 and IMP dehydrogenase 1 were down-regulated in the BAT of cold-exposed rats. The physiological meaning of these changes accompanying cold exposure was discussed.
Keywords: Brown adipose tissue (BAT); Microarray analysis; Gene expression;

Acknowledgement (113-114).