BBA - Bioenergetics (v.1506, #3)

Ultrafast haem–haem electron transfer in cytochrome c oxidase by Michael I. Verkhovsky; Audrius Jasaitis; Mårten Wikström (143-146).
Electron transfer between the redox centres is essential for the function of the haem–copper oxidases. To date, the fastest rate of electron transfer between the haem groups has been determined to be ca. 3×105 s−1. Here, we show by optical spectroscopy that about one half of this electron transfer actually occurs at least three orders of magnitude faster, after photolysis of carbon monoxide from the half-reduced bovine heart enzyme. We ascribe this to the true haem–haem electron tunnelling rate between the haem groups.
Keywords: electron tunnelling; CO photolysis; protein relaxation;

Comparative genomics and bioenergetics by Jose Castresana (147-162).
Bacterial and archaeal complete genome sequences have been obtained from a wide range of evolutionary lines, which allows some general conclusions about the phylogenetic distribution and evolution of bioenergetic pathways to be drawn. In particular, I searched in the complete genomes for key enzymes involved in aerobic and anaerobic respiratory pathways and in photosynthesis, and mapped them into an rRNA tree of sequenced species. The phylogenetic distribution of these enzymes is very irregular, and clearly shows the diverse strategies of energy conservation used by prokaryotes. In addition, a thorough phylogenetic analysis of other bioenergetic protein families of wide distribution reveals a complex evolutionary history for the respective genes. A parsimonious explanation for these complex phylogenetic patterns and for the irregular distribution of metabolic pathways is that the last common ancestor of Bacteria and Archaea contained several members of every gene family as a consequence of previous gene or genome duplications, while different patterns of gene loss occurred during the evolution of every gene family. This would imply that the last universal ancestor was a bioenergetically sophisticated organism. Finally, important steps that occurred during the evolution of energetic machineries, such as the early evolution of aerobic respiration and the acquisition of eukaryotic mitochondria from a proteobacterium ancestor, are supported by the analysis of the complete genome sequences.
Keywords: Genomics; Aerobic respiration; Anaerobic respiration; Photosynthesis; Last universal ancestor; Molecular evolution;

Properties of a proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli with α and β subunits linked through fused transmembrane helices by Johan Meuller; Kristin Mjörn; Jenny Karlsson; Anna Tigerström; Jan Rydström; Cynthia Hou; Philip D Bragg (163-171).
Proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli is composed of an α and a β subunit, whereas the homologues mitochondrial enzyme contains a single polypeptide. As compared to the latter transhydrogenase, using a 14-helix model for its membrane topology, the point of fusion is between the transmembrane helices 4 and 6 where the fusion linker provides the extra transmembrane helix 5. In order to clarify the potential role of this extra helix/linker, the α and the β subunits were fused using three connecting peptides of different lengths, one (pAX9) involving essentially a direct coupling, a second (pKM) with a linking peptide of 18 residues, and a third (pKMII) with a linking peptide of 32 residues, as compared to the mitochondrial extra peptide of 27 residues. The results demonstrate that the plasma membrane-bound and purified pAX9 enzyme with the short linker was partly misfolded and strongly inhibited with regard to both catalytic activities and proton translocation, whereas the properties of pKM and pKMII with longer linkers were similar to those of wild-type E. coli transhydrogenase but partly different from those of the mitochondrial enzyme although pKMII generally gave higher activities. It is concluded that a mitochondrial-like linking peptide is required for proper folding and activity of the E. coli fused transhydrogenase, and that differences between the catalytic properties of the E. coli and the mitochondrial enzymes are unrelated to the linking peptide. This is the first time that larger subunits of a membrane protein with multiple transmembrane helices have been fused with retained activity.
Keywords: Transhydrogenase; Nicotinamide adenine dinucleotide; Nicotinamide adenine dinucleotide, reduced; Proton pump; Membrane protein;

The chlorophyll a (Chla) fluorescence of cyanobacteria, which at physiological temperature originates from photosystem (PS) II holochromes, is suppressed in hyperosmotic suspension, and enhanced in hypo-osmotic suspension (G.C. Papageorgiou, A. Alygizaki-Zorba, Biochim. Biophys. Acta 1335 (1997) 1–4). We investigated the mechanism of this phenomenon by comparing Synechococcus sp. PCC 7942 cells that had been treated with N-ethylmaleimide (NEM) in order to inhibit electronic excitation transfers from phycobilisomes (PBS) to Chlas of PSI (A.N. Glazer, Y.M. Gindt, C.F. Chan, K. Sauer, Photosynth. Res. 40 (1994) 167–173) with untreated control cells. The NEM-treated cells were indistinguishable from the control cells with regard to PSII-dependent oxygen evolution, reduction of post-PSII oxidants, and osmotically induced volume changes, but differed in the following properties: (i) they could not photoreduce post-PSI electron acceptors; (ii) they diverted more PBS excitation to PSII; (iii) the rise of Chla fluorescence upon light acclimation of darkened (state 2) cells was smaller; and (iv) the Chla fluorescence of light-acclimated (state 1) cells was insensitive to the cell suspension osmolality. These properties suggest that osmolality regulates the core-mediated excitation coupling between PBS and PSI, possibly by influencing mutual orientation and/or distance between core holochromes (ApcE, ApcD) and PSI holochromes. Thus, in hyper-osmotic suspension, PBS deliver more excitation to PSI (hence less to PSII); in hypo-osmotic cell suspension they deliver less excitation to PSI (hence more to PSII).

The presence of 9-cis-β-carotene in cytochrome b 6 f complex from spinach by Jiusheng Yan; Yulong Liu; Dazhang Mao; Liangbi Li; Tingyun Kuang (182-188).
Cytochrome b 6 f complex with stoichiometrically bound β-carotene molecule was purified from spinach chloroplasts. The configuration of this β-carotene was studied by reversed-phase HPLC and resonance Raman spectroscopy. Both the absorption spectrum of this β-carotene in dissociated state and the Raman spectrum in native state can be unambiguously assigned to a 9-cis configuration. This finding is in contrast to the predominantly all-trans isomers commonly found in membranes and protein–pigment complexes of chloroplasts, suggesting that the 9-cis-β-carotene is an authentic component and may have a unique structural and functional role in cytochrome b 6 f complex.
Keywords: Cytochrome b 6 f; β-Carotene; Configuration; High performance liquid chromatography; Resonance Raman spectroscopy;

Coupling of proton flow to ATP synthesis in Rhodobacter capsulatus: F0F1-ATP synthase is absent from about half of chromatophores by Boris A Feniouk; Dmitry A Cherepanov; Wolfgang Junge; Armen Y Mulkidjanian (189-203).
F0F1-ATP synthase (H+-ATP synthase, F0F1) utilizes the transmembrane protonmotive force to catalyze the formation of ATP from ADP and inorganic phosphate (Pi). Structurally the enzyme consists of a membrane-embedded proton-translocating F0 portion and a protruding hydrophilic F1 part that catalyzes the synthesis of ATP. In photosynthetic purple bacteria a single turnover of the photosynthetic reaction centers (driven by a short saturating flash of light) generates protonmotive force that is sufficiently large to drive ATP synthesis. Using isolated chromatophore vesicles of Rhodobacter capsulatus, we monitored the flash induced ATP synthesis (by chemoluminescence of luciferin/luciferase) in parallel to the transmembrane charge transfer through F0F1 (by following the decay of electrochromic bandshifts of intrinsic carotenoids). With the help of specific inhibitors of F1 (efrapeptin) and of F0 (venturicidin), we decomposed the kinetics of the total proton flow through F0F1 into (i) those coupled to the ATP synthesis and (ii) the de-coupled proton escape through F0. Taking the coupled proton flow, we calculated the H+/ATP ratio; it was found to be 3.3±0.6 at a large driving force (after one saturating flash of light) but to increase up to 5.1±0.9 at a smaller driving force (after a half-saturating flash). From the results obtained, we conclude that our routine chromatophore preparations contained three subsets of chromatophore vesicles. Chromatophores with coupled F0F1 dominated in fresh material. Freezing/thawing or pre-illumination in the absence of ADP and Pi led to an increase in the fraction of chromatophores with at least one de-coupled F0(F1). The disclosed fraction of chromatophores that lacked proton-conducting F0(F1) (approx. 40% of the total amount) remained constant upon these treatments.
Keywords: Proton transfer; ATP synthesis; Electrochromism; Membrane potential,; Rhodobacter capsulatus;

Endothelial- and nitric oxide-dependent effects on oxidative metabolism of intact artery by John T Barron; Liping Gu; Joseph E Parrillo (204-211).
Oxidative metabolism and its possible modulation by nitric oxide (NO) was examined in endothelial-intact and endothelial-denuded segments of porcine carotid arteries. Endothelial-intact arteries displayed appropriate NO-mediated vasorelaxation to acetylcholine (ACh). Endothelial-denuded arteries demonstrated absent vasorelaxation to ACh stimulation and depressed contractile responsiveness to K+ depolarization, which was normalized by inhibition of NO synthesis by N ω-nitro-l-arginine methylester (l-NAME). Confirmation that carotid arteries continued to produce NO despite removal of the endothelium was indicated by detection of NO metabolites in the incubation medium bathing the arteries. O2 consumption and the oxidation of glucose and fatty acid were depressed in endothelial-denuded arteries. Depression of O2 consumption and glucose oxidation was completely reversed by treatment with l-NAME. We conclude that endogenous NO produced by non-endothelial vascular cells depresses contractility, O2 consumption, and oxidation of energy substrates in vascular smooth muscle. The endothelium may play a role in oxidative metabolism of vascular smooth muscle possibly by modulating the effects of NO produced by other cells of the vessel wall, or by other factors.
Keywords: Nitric oxide; Vascular smooth muscle; Oxidative metabolism; O2 consumption; Endothelium; Mitochondria;

Expression of sodium/proton antiporter NhaA at various pH values in Escherichia coli by Toshiaki Shijuku; Hiromi Saito; Tomohito Kakegawa; Hiroshi Kobayashi (212-217).
It was reported that NhaA, one of sodium/proton antiporters in Escherichia coli, was expressed at alkaline pH [J. Biol. Chem. 266 (1991) 21753]. In disagreement with their results, expression of an nhaA–lacZ fusion gene was found to be very low in an E. coli strain derived from MC4100 within the wide pH range from 5 to 9. When nhaB was deleted, the fusion gene was expressed at pH values below 8, while the expression was observed at alkaline pH after chaA was deleted. The internal level of sodium ions was increased by deletion of nhaA in strains deficient in nhaB and chaA at low and high pH values, respectively. These results suggested that nhaA is induced only when a low level of internal sodium ions is not kept by NhaB and ChaA. Strains used in the previous study may have low active ChaA.
Keywords: Na+/H+; nhaA; Antiporter; pH dependence; Gene expression; Escherichia coli;

Effect of deuterium oxide on actomyosin motility in vitro by Shigeru Chaen; Naoto Yamamoto; Ibuki Shirakawa; Haruo Sugi (218-223).
Actin filament velocities in an in vitro motility assay system were measured both in heavy water (deuterium oxide, D2O) and water (H2O) to examine the effect of D2O on the actomyosin interaction. The dependence of the sliding velocity on pD of the D2O assay solution showed a broad pD optimum of around pD 8.5 which resembled the broad pH optimum (pH 8.5) of the H2O assay solution, but the maximum velocity (4.1±0.5 μm/s, n=11) at pD 8.5 in D2O was about 60% of that (7.1±1.1 μm/s, n=11) at pH 8.5 in H2O. The K m values of 95 and 80 μM and V max values of 3.2 and 5.1 μm/s for the D2O and H2O assay were obtained by fitting the ATP concentration dependence of the velocity (at pD and pH 7.5) to the Michaelis–Menten equation. The K m value of actin-activated Mg-ATPase activity of myosin subfragment 1 (S1) was decreased from 50 μM [actin] in H2O to 33 μM [actin] in D2O without any significant changes in V max (9.4 s−1 in D2O and 9.3 s−1 in H2O). The rate constants of ADP release from the acto-S1–ADP complex measured by the stopped flow method were 361±26 s−1 (n=27) in D2O and 512±39 s−1 (n=27) in H2O at 6°C. These results suggest that the decrease in the in vitro actin-myosin sliding velocity in D2O results from a slowing of the release of ADP from the actomyosin–ADP complex and the increase in the affinity of actin for myosin in the presence of ATP in D2O.
Keywords: Deuterium oxide; Actomyosin; In vitro motility assay;

Photosynthetic water oxidation in Synechocystis sp. PCC6803: mutations D1-E189K, R and Q are without influence on electron transfer at the donor side of photosystem II by Jürgen Clausen; Stephanie Winkler; Anna-Maria A Hays; Monika Hundelt; Richard J Debus; Wolfgang Junge (224-235).
The oxygen-evolving manganese cluster (OEC) of photosynthesis is oxidised by the photochemically generated primary oxidant (P+⋅ 680) of photosystem II via a tyrosine residue (YZ, Tyr161 on the D1 subunit of Synechocystis sp. PCC6803). The redox span between these components is rather small and probably tuned by protonic equilibria. The very efficient electron transfer from YZ to P+⋅ 680 in nanoseconds requires the intactness of a hydrogen bonded network involving YZ, D1-His190, and presumably D1-Glu189. We studied photosystem II core particles from photoautotrophic mutants where the residue D1-E189 was replaced by glutamine, arginine and lysine which were expected to electrostatically differ from the glutamate in the wild-type (WT). Surprisingly, the rates of electron transfer from YZ to P+⋅ 680 as well as from the OEC to Yox Z were the same as in the WT. With the generally assumed proximity between D1-His190 (and thus D1-Glu189) and YZ, the lack of any influence on the electron transfer around YZ straightforwardly implies a strongly hydrophobic environment forcing Glu (acid) and Lys, Arg (basic) at position D1-189 into electro-neutrality. As one alternative, D1-Glu189 could be located at such a large distance from the OEC, YZ and P+⋅ 680 that a charge on D1-189X does not influence the electron transfer. This seems less likely in the light of the drastic influence of its direct neighbour, D1-His190, on YZ function. Another alternative is that D1-Glu189 is negatively charged, but is located in a cluster of acid/base groups that compensates for an alteration of charge at position 189, leaving the overall net charge unchanged in the Gln, Lys, and Arg mutants.
Keywords: Photosynthesis; Water oxidation; Proton coupled electron transfer; Tyrosine; Photosystem II;

Bicarbonate, an activating anion of ATP hydrolysis, inhibited ATP synthesis coupled to succinate oxidation in beef heart submitochondrial particles but diminished the lag time and increased the steady-state velocity of the 32Pi–ATP exchange reaction. The latter effects exclude the possibility that bicarbonate is inducing an intrinsic uncoupling between ATP hydrolysis and proton translocation at the level of F1Fo ATPase. The inhibition of ATP synthesis was competitive with respect to ADP at low fixed [Pi], mixed at high [Pi] and non-competitive towards Pi at any fixed [ADP]. From these results we can conclude that (i) bicarbonate does not bind to a Pi site in the mitochondrial F1; (ii) it competes with the binding of ADP to a low-affinity site, likely the low-affinity non-catalytic nucleotide binding site. It is postulated that bicarbonate stimulates ATP hydrolysis and inhibits ATP synthesis by modulating the relative affinities of the catalytic site for ATP and ADP.
Keywords: F1Fo ATP synthase; Mitochondrion; ATP synthesis; Inhibition; Activating anion; Bicarbonate;

A light-sensitive and chlorophyll (Chl)-deficient mutant of the green alga Dunaliella salina (dcd1) showed an amplified response to irradiance stress compared to the wild-type. The mutant was yellow–green under low light (100 μmol photons m−2 s−1) and yellow under high irradiance (2000 μmol photons m−2 s−1). The mutant had lower levels of Chl, lower levels of light harvesting complex II, and a smaller Chl antenna size. The mutant contained proportionately greater amounts of photodamaged photosystem (PS) II reaction centers in its thylakoid membranes, suggesting a greater susceptibility to photoinhibition. This phenotype was more pronounced under high than low irradiance. The Cbr protein, known to accumulate when D. salina is exposed to irradiance stress, was pronouncedly expressed in the mutant even under low irradiance. This positively correlated with a higher zeaxanthin content in the mutant. Cbr protein accumulation, xanthophyll cycle de-epoxidation state, and fraction of photodamaged PSII reaction centers in the thylakoid membrane showed a linear dependence on the chloroplast ‘photoinhibition index’, suggesting a cause-and-effect relationship between photoinhibition, Cbr protein accumulation and xanthophyll cycle de-epoxidation state. These results raised the possibility of zeaxanthin and Cbr involvement in the PSII repair process through photoprotection of the partially disassembled, and presumably vulnerable, PSII core complexes from potentially irreversible photooxidative bleaching.
Keywords: Chlorophyll antenna size; Photoinhibition; Zeaxanthin; Cbr protein; Photosystem II repair; Dunaliella salina;

In carotenoids the lowest energetic optical transition belonging to the π-electron system is forbidden by symmetry, therefore the energetic position of the S1 (21Ag) level can hardly be assessed by optical spectroscopy. We introduce a novel experimental approach: For molecules with π-electron systems the transition C1s→2p(π*) from inner-atomic to the lowest unoccupied molecular orbital (LUMO) appears in X-ray absorption near edge spectra (NEXAFS) as an intense, sharp peak a few eV below the carbon K-edge. Whereas the peak position reflects the energy of the first excited singlet state in relation to the ionization potential of the molecule, intensity and width of the transition depend on hybridization and bonding partners of the selected atom. Complementary information can be obtained from ultraviolet photoelectron spectroscopy (UPS): At the low binding energy site of the spectrum a peak related to the highest occupied molecular orbital (HOMO) appears. We have measured NEXAFS and UPS of β-carotene. Based on these measurements and quantum chemical calculations the HOMO and LUMO energies can be derived.
Keywords: NEXAFS; UPS; β-carotene; HOMO; LUMO; S1;

Author Index (268-269).

Cumulative Contents (270-271).