BBA - Bioenergetics (v.1797, #9)
Editorial Board (i).
Electron flow through metalloproteins by Harry B. Gray; Jay R. Winkler (1563-1572).
Electron transfers in photosynthesis and respiration commonly occur between metal-containing cofactors that are separated by large molecular distances. Understanding the underlying physics and chemistry of these biological electron transfer processes is the goal of much of the work in our laboratories. Employing laser flash-quench triggering methods, we have shown that 20 Å, coupling-limited Fe(II) to Ru(III) and Cu(I) to Ru(III) electron tunneling in Ru-modified cytochromes and blue copper proteins can occur on the microsecond timescale both in solutions and crystals; and, further, that analysis of these rates suggests that distant donor–acceptor electronic couplings are mediated by a combination of sigma and hydrogen bonds in folded polypeptide structures. Redox equivalents can be transferred even longer distances by multistep tunneling, often called hopping, through intervening amino acid side chains. In recent work, we have found that 20 Å hole hopping through an intervening tryptophan is several hundred-fold faster than single-step electron tunneling in a Re-modified blue copper protein.
Keywords: Electron transfer; Cytochrome c; Azurin; Cytochrome b 562; Electron tunneling; Electron hopping;
Guidelines for tunneling in enzymes by Christopher C. Moser; J.L. Ross Anderson; P. Leslie Dutton (1573-1586).
Here we extend the engineering descriptions of simple, single-electron-tunneling chains common in oxidoreductases to quantify sequential oxidation–reduction rates of two-or-more electron cofactors and substrates. We identify when nicotinamides may be vulnerable to radical mediated oxidation–reduction and merge electron-tunneling expressions with the chemical rate expressions of Eyring. The work provides guidelines for the construction of new artificial oxidoreductases inspired by Nature but adopting independent design and redox engineering.
Keywords: Electron tunneling; Hydride transfer; Marcus theory; Protein engineering;
Occurrence, biosynthesis and function of isoprenoid quinones by Beatrycze Nowicka; Jerzy Kruk (1587-1605).
Isoprenoid quinones are one of the most important groups of compounds occurring in membranes of living organisms. These compounds are composed of a hydrophilic head group and an apolar isoprenoid side chain, giving the molecules a lipid-soluble character. Isoprenoid quinones function mainly as electron and proton carriers in photosynthetic and respiratory electron transport chains and these compounds show also additional functions, such as antioxidant function. Most of naturally occurring isoprenoid quinones belong to naphthoquinones or evolutionary younger benzoquinones. Among benzoquinones, the most widespread and important are ubiquinones and plastoquinones. Menaquinones, belonging to naphthoquinones, function in respiratory and photosynthetic electron transport chains of bacteria. Phylloquinone K1, a phytyl naphthoquinone, functions in the photosynthetic electron transport in photosystem I. Ubiquinones participate in respiratory chains of eukaryotic mitochondria and some bacteria. Plastoquinones are components of photosynthetic electron transport chains of cyanobacteria and plant chloroplasts. Biosynthetic pathway of isoprenoid quinones has been described, as well as their additional, recently recognized, diverse functions in bacterial, plant and animal metabolism.►Isoprenoid quinones are found in nearly all living organisms. ►Isoprenoid quinones belong tomenaquinones, ubiquinones or plastoquinones. ►Isoprenoid quinones function in electron transport chains and are antioxidants. ►Menaquinones in prokaryotes are cofactors of many enzymes. ►Biosynthesis of various isoprenoid quinones is well characterized.
Keywords: Biosynthesis; Electron transport chain; Isoprenoid quinone; Naphthoquinone; Plastoquinone; Ubiquinone;
Energy transfer processes in the isolated core antenna complexes CP43 and CP47 of photosystem II by Anna Paola Casazza; Malwina Szczepaniak; Marc G. Müller; Giuseppe Zucchelli; Alfred R. Holzwarth (1606-1616).
The energy equilibration and transfer processes in the isolated core antenna complexes CP43 and CP47 of photosystem II have been studied by steady-state and ultrafast (femto- to nanosecond) time-resolved spectroscopy at room temperature. The annihilation-free femtosecond absorption data can be described by surprisingly simple sequential kinetic models, in which the excitation energy transfer between blue and red states in both antenna complexes is dominated by sub-picosecond processes and is completed in less than 2 ps. The slowest energy transfer steps with lifetimes in the range of 1–2 ps are assigned to transfer steps between the chlorophyll layers located on the stromal and lumenal sides. We conclude that these ultrafast intra-antenna energy transfer steps do not represent a bottleneck in the rate of the primary processes in intact photosystem II. Since the experimental energy equilibration rates are up to a factor of 3–5 higher than concluded previously, our results challenge the conclusions drawn from theoretical modeling.
Keywords: antenna complexes; photosystem II; energy transfer; fluorescence kinetics; femtosecond absorption; photosynthesis;
Electronic structure of the primary electron donor of Blastochloris viridis heterodimer mutants: High-field EPR study by N.S. Ponomarenko; O.G. Poluektov; E.J. Bylina; J.R. Norris (1617-1626).
High-field electron paramagnetic resonance (HF EPR) has been employed to investigate the primary electron donor electronic structure of Blastochloris viridis heterodimer mutant reaction centers (RCs). In these mutants the amino acid substitution His(M200)Leu or His(L173)Leu eliminates a ligand to the primary electron donor, resulting in the loss of a magnesium in one of the constituent bacteriochlorophylls (BChl). Thus, the native BChl/BChl homodimer primary donor is converted into a BChl/bacteriopheophytin (BPhe) heterodimer. The heterodimer primary donor radical in chemically oxidized RCs exhibits a broadened EPR line indicating a highly asymmetric distribution of the unpaired electron over both dimer constituents. Observed triplet state EPR signals confirm localization of the excitation on the BChl half of the heterodimer primary donor. Theoretical simulation of the triplet EPR lineshapes clearly shows that, in the case of mutants, triplet states are formed by an intersystem crossing mechanism in contrast to the radical pair mechanism in wild type RCs. Photooxidation of the mutant RCs results in formation of a BPhe anion radical within the heterodimer pair. The accumulation of an intradimer BPhe anion is caused by the substantial loss of interaction between constituents of the heterodimer primary donor along with an increase in the reduction potential of the heterodimer primary donor D/D+ couple. This allows oxidation of the cytochrome even at cryogenic temperatures and reduction of each constituent of the heterodimer primary donor individually. Despite a low yield of primary donor radicals, the enhancement of the semiquinone–iron pair EPR signals in these mutants indicates the presence of kinetically viable electron donors.
Keywords: Blastochloris viridis; Heterodimer mutant; High-field EPR; Triplet state; Heterodimer; Photosynthetic reaction center; Cytochrome; Electron transfer;
Extracellular calcium depletion transiently elevates oxygen consumption in neurosecretory PC12 cells through activation of mitochondrial Na+/Ca2+ exchange by Alexander V. Zhdanov; Manus W. Ward; Cormac T. Taylor; Ekaterina A. Souslova; Dmitri M. Chudakov; Jochen H.M. Prehn; Dmitri B. Papkovsky (1627-1637).
Fluctuating extracellular Ca2+ regulates many aspects of neuronal (patho)physiology including cell metabolism and respiration. Using fluorescence-based intracellular oxygen sensing technique, we demonstrate that depletion of extracellular Ca2+ from 1.8 to ≤ 0.6 mM by chelation with EGTA induces a marked spike in O2 consumption in differentiated PC12 cells. This respiratory response is associated with the reduction in cytosolic and mitochondrial Ca2+, minor depolarization on the mitochondrial membrane, moderate depolarization of plasma membrane, and no changes in NAD(P)H and ATP. The response is linked to the influx of extracellular Na+ and the subsequent activation of mitochondrial Na+/Ca2+ and Na+/H+ exchange. The mitochondrial Na+/Ca2+ exchanger (mNCX) activated by Na+ influx reduces Ca2+ and increases Na+ levels in the mitochondrial matrix. The excess of Na+ activates the mitochondrial Na+/H+ exchanger (NHE) increasing the outward pumping of protons, electron transport and O2 consumption. Reduction in extracellular Na+ and inhibition of Na+ influx through the receptor operated calcium channels and plasmalemmal NHE reduce the respiratory response. Inhibition of the mNCX, L-type voltage gated Ca2+ channels or the release of Ca2+ from the endoplasmic reticulum also reduces the respiratory spike, indicating that unimpaired intercompartmental Ca2+ exchange is critical for response development.
Keywords: Extracellular calcium; Intracellular calcium; Cell Metabolism; Respiration; Mitochondria; Phosphorescent Oxygen-Sensitive Probe;
Role of specific residues in coenzyme binding, charge–transfer complex formation, and catalysis in Anabaena ferredoxin NADP+-reductase by José Ramón Peregrina; Ana Sánchez-Azqueta; Beatriz Herguedas; Marta Martínez-Júlvez; Milagros Medina (1638-1646).
Two transient charge–transfer complexes (CTC) form prior and upon hydride transfer (HT) in the reversible reaction of the FAD-dependent ferredoxin-NADP+ reductase (FNR) with NADP+/H, FNRox-NADPH (CTC-1), and FNRrd-NADP+ (CTC-2). Spectral properties of both CTCs, as well as the corresponding interconversion HT rates, are here reported for several Anabaena FNR site-directed mutants. The need for an adequate initial interaction between the 2′P-AMP portion of NADP+/H and FNR that provides subsequent conformational changes leading to CTC formation is further confirmed. Stronger interactions between the isoalloxazine and nicotinamide rings might relate with faster HT processes, but exceptions are found upon distortion of the active centre. Thus, within the analyzed FNR variants, there is no strict correlation between the stability of the transient CTCs formation and the rate of the subsequent HT. Kinetic isotope effects suggest that, while in the WT, vibrational enhanced modulation of the active site contributes to the tunnel probability of HT; complexes of some of the active site mutants with the coenzyme hardly allow the relative movement of isoalloxazine and nicotinamide rings along the HT reaction. The architecture of the WT FNR active site precisely contributes to reduce the stacking probability between the isoalloxazine and nicotinamide rings in the catalytically competent complex, modulating the angle and distance between the N5 of the FAD isoalloxazine and the C4 of the coenzyme nicotinamide to values that ensure efficient HT processes.
Keywords: Ferredoxin-NADP+ reductase; Hydride transfer; Charge–transfer complex; Site-directed mutagenesis; Kinetic isotopic effect; Isoalloxazine–nicotinamide interactions;
Pigment organization in fucoxanthin chlorophyll a/c2 proteins (FCP) based on resonance Raman spectroscopy and sequence analysis by Lavanya Premvardhan; Bruno Robert; Anja Beer; Claudia Büchel (1647-1656).
Chlorophylls (Chls)-a and -c2 are identified and characterized in fucoxanthin chlorophyll-a/c2 protein (FCP) complexes in the trimeric (FCPatrim) and oligomeric (FCPbolig) forms of FCP from the diatom Cyclotella meneghiniana using resonance Raman (RR) spectroscopy. Importantly, two different Chl-c2s are identified in both FCPatrim and FCPbolig from their signature ring-breathing modes at ∼ 1360 cm− 1. In addition, the C131-keto carbonyl peaks indicate the presence of more than four Chl-a's in both FCP complexes and are broadly classified into three groups with strong, medium and weak external hydrogen bonds. Together, they provide the strongest spectroscopic evidence so far that there may be up to double the number of pigments previously estimated at 4Fx:4Chl-a:1Chl-c2 per FCP monomer. Careful analysis of the protein sequences also strongly support the higher pigment content by showing that at least six Chl-a, and one Chl-b, binding sites found in LHCII are retained in the FCPs. The relative enhancement of the RR bands for 406.7 versus 413.1 nm further allows some distinction of blue- versus red-absorbing Chl-a’s, respectively. Further differences between the Chls in FCPbolig and FCPatrim are present in the amino-acid sequences and the RR signals. Information about the Chl-binding sites, complemented by information about the structures and interactions of the Chls are used to characterize their local environments, and assign pigment locations (and functions) in FCPbolig and FCPatrim, which along with the earlier characterization of the carotenoids (J. Phys. Chem. B. 112 (2009) 12565–12574) provide a first (global) framework for pigment organization in FCP.
Keywords: Diatoms; Light-harvesting complex; Chlorophyll structure; Pigment organization;
Heme–heme and heme–ligand interactions in the di-heme oxygen-reducing site of cytochrome bd from Escherichia coli revealed by nanosecond absorption spectroscopy by Fabrice Rappaport; Jie Zhang; Marten H. Vos; Robert B. Gennis; Vitaliy B. Borisov (1657-1664).
Cytochrome bd is a terminal quinol:O2 oxidoreductase of respiratory chains of many bacteria. It contains three hemes, b 558, b 595, and d. The role of heme b 595 remains obscure. A CO photolysis/recombination study of the membranes of Escherichia coli containing either wild type cytochrome bd or inactive E445A mutant was performed using nanosecond absorption spectroscopy. We compared photoinduced changes of heme d–CO complex in one-electron-reduced, two-electron-reduced, and fully reduced states of cytochromes bd. The line shape of spectra of photodissociation of one-electron-reduced and two-electron-reduced enzymes is strikingly different from that of the fully reduced enzyme. The difference demonstrates that in the fully reduced enzyme photolysis of CO from heme d perturbs ferrous heme b 595 causing loss of an absorption band centered at 435 nm, thus supporting interactions between heme b 595 and heme d in the di-heme oxygen-reducing site, in agreement with previous works. Photolyzed CO recombines with the fully reduced enzyme monoexponentially with τ ∼ 12 μs, whereas recombination of CO with one-electron-reduced cytochrome bd shows three kinetic phases, with τ ∼ 14 ns, 14 μs, and 280 μs. The spectra of the absorption changes associated with these components are different in line shape. The 14 ns phase, absent in the fully reduced enzyme, reflects geminate recombination of CO with part of heme d. The 14-μs component reflects bimolecular recombination of CO with heme d and electron backflow from heme d to hemes b in ∼ 4% of the enzyme population. The final, 280-μs component, reflects return of the electron from hemes b to heme d and bimolecular recombination of CO in that population. The fact that even in the two-electron-reduced enzyme, a nanosecond geminate recombination is observed, suggests that namely the redox state of heme b 595, and not that of heme b 558, controls the pathway(s) by which CO migrates between heme d and the medium.
Keywords: Respiration; Chlorin; Cytochrome; Ligand binding; Gas molecule; Photobiology;
Determination of the intrinsic redox potentials of FeS centers of respiratory complex I from experimental titration curves by Emile S. Medvedev; Vernon A. Couch; Alexei A. Stuchebrukhov (1665-1671).
Recently, Euro et al. [Biochem. 47, 3185 (2008) ] have reported titration data for seven of nine FeS redox centers of complex I from Escherichia coli. There is a significant uncertainty in the assignment of the titration data. Four of the titration curves were assigned to N1a, N1b, N6b, and N2 centers; one curve either to N3 or N7; one more either to N4 or N5; and the last one denoted Nx could not be assigned at all. In addition, the assignment of the titration data to the N6b/N6a pair is also uncertain. In this paper, using our calculated interaction energies [Couch et al. BBA 1787, 1266 (2009)], we perform statistical analysis of these data, considering a variety of possible assignments, find the best fit, and determine the intrinsic redox potentials of the centers. The intrinsic potentials could be determined with an uncertainty of less than ± 10 mV at a 95% confidence level for best fit assignments. We also find that the best agreement between theoretical and experimental titration curves is obtained with the N6b–N2 interaction equal to 71 ± 14 or 96 ± 26 mV depending on the N6b/N6a titration data assignment, which is stronger than was expected and may indicate a close distance of the N2 center to the membrane surface.
Keywords: Complex I; Electron transfer;
Mitochondrial respiration and membrane potential are regulated by the allosteric ATP-inhibition of cytochrome c oxidase by Rabia Ramzan; Katrin Staniek; Bernhard Kadenbach; Sebastian Vogt (1672-1680).
This paper describes the problems of measuring the allosteric ATP-inhibition of cytochrome c oxidase (CcO) in isolated mitochondria. Only by using the ATP-regenerating system phosphoenolpyruvate and pyruvate kinase full ATP-inhibition of CcO could be demonstrated by kinetic measurements. The mechanism was proposed to keep the mitochondrial membrane potential (∆Ψm) in living cells and tissues at low values (100–140 mV), when the matrix ATP/ADP ratios are high. In contrast, high ∆Ψm values (180–220 mV) are generally measured in isolated mitochondria. By using a tetraphenyl phosphonium electrode we observed in isolated rat liver mitochondria with glutamate plus malate as substrates a reversible decrease of ∆Ψm from 233 to 123 mV after addition of phosphoenolpyruvate and pyruvate kinase. The decrease of ∆Ψm is explained by reversal of the gluconeogenetic enzymes pyruvate carboxylase and phosphoenolpyruvate carboxykinase yielding ATP and GTP, thus increasing the matrix ATP/ADP ratio. With rat heart mitochondria, which lack these enzymes, no decrease of ∆Ψm was found. From the data we conclude that high matrix ATP/ADP ratios keep ∆Ψm at low values by the allosteric ATP-inhibition of CcO, thus preventing the generation of reactive oxygen species which could generate degenerative diseases. It is proposed that respiration in living eukaryotic organisms is normally controlled by the ∆Ψm-independent “allosteric ATP-inhibition of CcO.” Only when the allosteric ATP-inhibition is switched off under stress, respiration is regulated by “respiratory control,” based on ∆Ψm according to the Mitchell Theory.► Regulation of mitochondrial respiration. ► Regulation of mitochondrial membrane potential. ► Regulation of Cytochrome c oxidase activity. ► Phosphorylation of Cytochrome c oxidase subunits.
Keywords: Cytochrome c oxidase; Regulation of respiration; Mitochondrial membrane potential; ATP/ADP ratio; Respiratory control; Tetraphenyl phosphonium electrode;
Possibilities of subunit localization with fluorescent protein tags and electron microscopy examplified by a cyanobacterial NDH-1 study by Mariam Birungi; Mihaela Folea; Natalia Battchikova; Min Xu; Hualing Mi; Teruo Ogawa; Eva-Mari Aro; Egbert J. Boekema (1681-1686).
Cyanobacterial NDH-1 is a multisubunit complex involved in proton translocation, cyclic electron flow around photosystem I and CO2 uptake. The function and location of several of its small subunits are unknown. In this work, the location of the small subunits NdhL, -M, -N, -O and CupS of Synechocystis 6803 NDH-1 was established by electron microscopy (EM) and single particle analysis. To perform this, the subunits were enlarged by fusion with the YFP protein. After classification of projections, the position of the YFP tag was revealed; all five subunits are integrated in the membrane domain. The results on NDH-1 demonstrate that a GFP tag can be revealed after data processing of EM data sets of moderate size, thus showing that this way of labeling is a fast and reliable way for subunit mapping in multisubunit complexes after partial purification.
Keywords: NDH-1; Electron microscopy; Subunit labeling; Yellow fluorescent protein; Synechocystis 6803;