Mutations in cytochrome b that affect kinetics of the electron transfer reactions at center N in the yeast cytochrome bc1 complex

We have examined the pre-steady-state kinetics and thermodynamic properties of the b hemes in variants of the yeast cytochrome bc₁ complex that have mutations in the quinone reductase site (center N). Trp-30 is a highly conserved residue, forming a hydrogen bond with the propionate on the high potential b heme (b_H heme). The substitution by a cysteine (W30C) lowers the redox potential of the heme and an apparent consequence is a lower rate of electron transfer between quinol and heme at center N. Leu-198 is also in close proximity to the b_H heme and a L198F mutation alters the spectral properties of the heme but has only minor effects on its redox properties or the electron transfer kinetics at center N. Substitution of Met-221 by glutamine or glutamate results in the loss of a hydrophobic interaction that stabilizes the quinone ligands. Ser-20 and Gln-22 form a hydrogen-bonding network that includes His-202, one of the carbonyl groups of the ubiquinone ring, and an active-site water. A S20T mutation has long-range structural effects on center P and thermodynamic effects on both b hemes. The other mutations (M221E, M221Q, Q22E and Q22T) do not affect the ubiquinol oxidation kinetics at center P, but do modify the electron transfer reactions at center N to various extents. The pre-steady reduction kinetics suggest that these mutations alter the binding of quinone ligands at center N, possibly by widening the binding pocket and thus increasing the distance between the substrate and the b_H heme. These results show that one can distinguish between the contribution of structural and thermodynamic factors to center N function.     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

Modelling of the electron transfer reactions in Photosystem I by electron tunnelling theory: The phylloquinones bound to the PsaA and the PsaB reaction centre subunits of PS I are almost isoenergetic to the iron-sulfur cluster FX

Photosystem I is a large macromolecular complex located in the thylakoid membranes of chloroplasts and in cyanobacteria that catalyses the light driven reduction of ferredoxin and oxidation of plastocyanin. Due to the very negative redox potential of the primary electron transfer cofactors accepting electrons, direct estimation by redox titration of the energetics of the system is hampered. However, the rates of electron transfer reactions are related to the thermodynamic properties of the system. Hence, several spectroscopic and biochemical techniques have been employed, in combination with the classical Marcus theory for electron transfer tunnelling, in order to access these parameters. Nevertheless, the values which have been presented are very variable. In particular, for the case of the tightly bound phylloquinone molecule A₁, the values of the redox potentials reported in the literature vary over a range of about 350 mV. Previous models of Photosystem I have assumed a unidirectional electron transfer model. In the present study, experimental evidence obtained by means of time resolved absorption, photovoltage, and electron paramagnetic resonance measurements are reviewed and analysed in terms of a bi-directional kinetic model for electron transfer reactions. This model takes into consideration the thermodynamic equilibrium between the iron-sulfur centre F_X and the phylloquinone bound to either the PsaA (A_1A) or the PsaB (A_1B) subunit of the reaction centre and the equilibrium between the iron-sulfur centres F_A and F_B. The experimentally determined decay lifetimes in the range of sub-picosecond to the microsecond time domains can be satisfactorily simulated, taking into consideration the edge-to-edge distances between redox cofactors and driving forces reported in the literature. The only exception to this general behaviour is the case of phylloquinone (A₁) reoxidation. In order to describe the reported rates of the biphasic decay, of about 20 and 200 ns, associated with this electron transfer step, the redox potentials of the quinones are estimated to be almost isoenergetic with that of the iron sulfur centre F_X. A driving force in the range of 5 to 15 meV is estimated for these reactions, being slightly exergonic in the case of the A_1B quinone and slightly endergonic, in the case of the A_1A quinone. The simulation presented in this analysis not only describes the kinetic data obtained for the wild type samples at room temperature and is consistent with estimates of activation energy by the analysis of temperature dependence, but can also explain the effect of the mutations around the PsaB quinone binding pocket. A model of the overall energetics of the system is derived, which suggests that the only substantially irreversible electron transfer reactions are the reoxidation of A₀ on both electron transfer branches and the reduction of F_A by F_X.     

Time-resolved OH → EH transition of the aberrant ba3 oxidase from Thermus thermophilus

The kinetics of single-electron injection into the oxidized nonrelaxed state (O_H → E_H transition) of the aberrant ba₃ cytochrome oxidase from Thermus thermophilus, noted for its lowered efficiency of proton pumping, was investigated by time-resolved optical spectroscopy. Two main phases of intraprotein electron transfer were resolved. The first component (τ ∼ 17 μs) reflects oxidation of Cu_A and reduction of the heme groups (low-spin heme b and high-spin heme a₃ in a ratio close to 50:50). The subsequent component (τ ∼ 420 μs) includes reoxidation of both hemes by Cu_B. This is in significant contrast to the O_H → E_H transition of the aa₃-type cytochrome oxidase from Paracoccus denitrificans, where the fastest phase is exclusively due to transient reduction of the low-spin heme a, without electron equilibration with the binuclear center. On the other hand, the one-electron reduction of the relaxed O state in ba₃ oxidase was similar to that in aa₃ oxidase and only included rapid electron transfer from Cu_A to the low-spin heme b. This indicates a functional difference between the relaxed O and the pulsed O_H forms also in the ba₃ oxidase from T. thermophilus.     

Function of two β-carotenes near the D1 and D2 proteins in photosystem II dimers

The antenna proteins in photosystem II (PSII) not only promote energy transfer to the photosynthetic reaction center (RC) but provide also an efficient cation sink to re-reduce chlorophyll a if the electron transfer (ET) from the Mn-cluster is inhibited. Using the newest PSII dimer crystal structure (3.0 Å resolution), in which 11 β-carotene molecules (Car) and 14 lipids are visible in the PSII monomer, we calculated the redox potentials (E_m) of one-electron oxidation for all Car (E_m(Car)) by solving the Poisson-Boltzmann equation. In each PSII monomer, the D1 protein harbors a previously unlocated Car (Car_D1) in van der Waals contact with the chlorin ring of Chl_Z(D1). Each Car_D1 in the PSII dimer complex is located in the interface between the D1 and CP47 subunits, together with another four Car of the other PSII monomer and several lipid molecules. The proximity of Car bridging between Car_D1 and plastoquinone/Q_A may imply a direct charge recombination of Car⁺Q_A⁻. The calculated E_m(Car_D1) and E_m(Chl_Z(D1)) are, respectively, 83 and 126 mV higher than E_m(Car_D2) and E_m(Chl_Z(D2)), which could explain why Car_D2⁺ and Chl_Z(D2)⁺ are observed rather than the corresponding Car_D1⁺ and Chl_Z(D1)⁺.     

Interaction of the Thermoplasma acidophilum A1A0-ATP synthase peripheral stalk with the catalytic domain

The peripheral stalk of the archaeal ATP synthase (A₁A₀)-ATP synthase is formed by the heterodimeric EH complex and is part of the stator domain, which counteracts the torque of rotational catalysis. Here we used nuclear magnetic resonance spectroscopy to probe the interaction of the C-terminal domain of the EH heterodimer (E_CT1H_CT) with the N-terminal 23 residues of the B subunit (B_NT). The data show a specific interaction of B_NT peptide with 26 residues of the E_CT1H_CT domain, thereby providing a molecular picture of how the peripheral stalk is anchored to the A₃B₃ catalytic domain in A₁A₀.

Structured summary

MINT-7260681: Hct (refseq:NP_393485), Ect1 (uniprotkb:Q9HM68) and Bnt (uniprotkb:Q9HM64) physically interact (MI:0915) by nuclear magnetic resonance (MI:0077)     

Synthesis, biological activity and molecular modelling studies of tricyclic alkylimidazo-, pyrimido- and diazepinopurinediones

Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5–35) were initially evaluated for their affinity at rat A₁ and A_2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (K_i = 0.28 μM) with fivefold A_2A selectivity and the non-selective A₁/A_2A AR ligand pyrimidopurinedione 35 (K_i A₁ = 0.28 μM and K_i A_2A = 0.30 μM). The compounds were also evaluated for their affinity at human A₁, A_2A, A_2B and A₃ ARs. All of the obtained compounds were docked to the A_2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist—XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A_2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds.

Electronic supplementary material

The online version of this article (doi:10.1007/s11302-013-9358-3) contains supplementary material, which is available to authorized users.     

Direct and mediated electron transfer between intact succinate:quinone oxidoreductase from Bacillus subtilis and a surface modified gold electrode reveals redox state-dependent conformational changes

Succinate:quinone oxidoreductase (SQR) from Bacillus subtilis consists of two hydrophilic protein subunits comprising succinate dehydrogenase, and a di-heme membrane anchor protein harboring two putative quinone binding sites, Q_p and Q_d. In this work we have used spectroelectrochemistry to study the electronic communication between purified SQR and a surface modified gold capillary electrode. In the presence of two soluble quinone mediators the midpoint potentials of both hemes were revealed essentially as previously determined by conventional redox titration (heme b_H, E_m = + 65 mV, heme b_L, E_m = − 95 mV). In the absence of mediators the enzyme still communicated with the electrode, albeit with a reproducible hysteresis, resulting in the reduction of both hemes occurring approximately at the midpoint potential of heme b_L, and with a pronounced delay of reoxidation. When the specific inhibitor 2-n-heptyl-4 hydroxyquinoline N-oxide (HQNO), which binds to Q_d in B. subtilis SQR, was added together with the two quinone mediators, rapid reductive titration was still possible which can be envisioned as an electron transfer occurring via the HQNO insensitive Q_p site. In contrast, the subsequent oxidative titration was severely hampered in the presence of HQNO, in fact it completely resembled the unmediated reaction. If mediators communicate with Q_p or Q_d, either event is followed by very rapid electron redistribution within the enzyme. Taken together, this strongly suggests that the accessibility of Q_p depended on the redox state of the hemes. When both hemes were reduced, and Q_d was blocked by HQNO, quinone-mediated communication via the Q_p site was no longer possible, revealing a redox-dependent conformational change in the membrane anchor domain.     

Spectroscopic studies of the chlorophyll d containing photosystem I from the cyanobacterium, Acaryochloris marina

Absorbance difference spectroscopy and redox titrations have been applied to investigate the properties of photosystem I from the chlorophyll d containing cyanobacterium Acaryochloris marina. At room temperature, the (P740⁺ − P740) and (F_A/B⁻ − F_A/B) absorbance difference spectra were recorded in the range between 300 and 1000 nm while at cryogenic temperatures, (P740⁺A₁⁻ − P740A₁) and (³P740 − P740) absorbance difference spectra have been measured. Spectroscopic and kinetic evidence is presented that the cofactors involved in the electron transfer from the reduced secondary electron acceptor, phylloquinone (A₁⁻), to the terminal electron acceptor and their structural arrangement are virtually identical to those of chlorophyll a containing photosystem I. The oxidation potential of the primary electron donor P740 of photosystem I has been reinvestigated. We find a midpoint potential of 450 ± 10 mV in photosystem I-enriched membrane fractions as well as in thylakoids which is very similar to that found for P700 in chlorophyll a dominated organisms. In addition, the extinction difference coefficient for the oxidation of the primary donor has been determined and a value of 45,000 ± 4000 M^− 1 cm^− 1 at 740 nm was obtained. Based on this value the ratio of P740 to chlorophyll is calculated to be 1:~ 200 chlorophyll d in thylakoid membranes. The consequences of our findings for the energetics in photosystem I of A. marina are discussed as well as the pigment stoichiometry and spectral characteristics of P740.     

Regulatory interactions in the dimeric cytochrome bc1 complex: The advantages of being a twin

The dimeric cytochrome bc₁ complex catalyzes the oxidation-reduction of quinol and quinone at sites located in opposite sides of the membrane in which it resides. We review the kinetics of electron transfer and inhibitor binding that reveal functional interactions between the quinol oxidation site at center P and quinone reduction site at center N in opposite monomers in conjunction with electron equilibration between the cytochrome b subunits of the dimer. A model for the mechanism of the bc₁ complex has emerged from these studies in which binding of ligands that mimic semiquinone at center N regulates half-of-the-sites reactivity at center P and binding of ligands that mimic catalytically competent binding of ubiquinol at center P regulates half-of-the-sites reactivity at center N. An additional feature of this model is that inhibition of quinol oxidation at the quinone reduction site is avoided by allowing catalysis in only one monomer at a time, which maximizes the number of redox acceptor centers available in cytochrome b for electrons coming from quinol oxidation reactions at center P and minimizes the leakage of electrons that would result in the generation of damaging oxygen radicals.     

Cyanobacterial cytochrome cM: Probing its role as electron donor for CuA of cytochrome c oxidase

It is well known that efficient functioning of photosynthetic (PET) and respiratory electron transport (RET) in cyanobacteria requires the presence of either cytochrome c₆ (Cytc₆) or plastocyanin (PC). By contrast, the interaction of an additional redox carrier, cytochrome c_M (Cytc_M), with either PET or RET is still under discussion. Here, we focus on the (putative) role of Cytc_M in cyanobacterial respiration. It is demonstrated that genes encoding the main terminal oxidase (cytochrome c oxidase, COX) and cytochrome c_M are found in all 44 totally or partially sequenced cyanobacteria (except one strain). In order to check whether Cytc_M can act as electron donor to COX, we investigated the intermolecular electron transfer kinetics between Cytc_M and the soluble Cu_A domain (i.e. the donor binding and electron entry site) of subunit II of COX. Both proteins from Synechocystis PCC6803 were expressed heterologously in E. coli. The forward and the reverse electron transfer reactions were studied yielding apparent bimolecular rate constants of (2.4 ± 0.1) × 10⁵ M^− 1 s^− 1 and (9.6 ± 0.4) × 10³ M^− 1 s^− 1 (5 mM phosphate buffer, pH 7, 50 mM KCl). A comparative analysis with Cytc₆ and PC demonstrates that Cytc_M functions as electron donor to Cu_A as efficiently as Cytc₆ but more efficient than PC. Furthermore, we demonstrate the association of Cytc_M with the cytoplasmic and thylakoid membrane fractions by immunobloting and discuss the potential role of Cytc_M as electron donor for COX under stress conditions.     

Involvement of plastoquinone bound within the isolated cytochrome b6-f complex from chloroplasts in oxidant-induced reduction of cytochrome b6

(1) Oxidant-induced reduction of cytochrome b₆ is completely dependent on a reduced component within the isolated cytochrome b₆-f complex. This component can be reduced by dithionite or by NADH/N-methylphenazonium methosulfate. It is a 2H⁺/2e⁻ carrier with a midpoint potential of 100 mV at pH 7.0, which is very similar to the midpoint potential of the plastoquinone pool in chloroplasts. (2) Oxidant-induced reduction of cytochrome b₆ is stimulated by plastoquinol-1 as well as by plastoquinol-9. The midpoint potential of the transient reduction of cytochrome b₆, however, was not shifted by added plastoquinol. (3) Quinone analysis of the purified cytochrome b₆-f complex revealed about one plastoquinone per cytochrome f. The endogenous quinone is heterogeneous, a form more polar than plastoquinone-A, probably plastoquinone-C, dominating, This is different from the thylakoid membrane where plastoquinone-A is the main quinone. (4) The endogenous quinone can be extracted from the lyophilized cytochrome b₆-f complex by acetone, but not by hydrocarbon solvents. Oxidant-induced reduction of cytochrome b₆ was observed in the lyophilized and hexane-extracted complex, but was lost in the acetone-extracted complex. Reconstitution was achieved either with plastoquinol-1 or plastoquinol-9, suggesting that a plastoquinol molecule is involved in oxidant-induced reduction of cytochrome b₆.     

Biosynthetic origin of 2-geranyl-1,4-naphthoquinone and its related anthraquinone in a Sesamum indicum hairy root culture

In order to clarify the biosynthetic origin of 2-geranyl-1,4-naphthoquinone and its biogenetically related anthraquinone, which are possible intermediates of anthrasesamones, [1-¹³C]glucose was administered to a hairy root culture of Sesamum indicum. The labeling patterns of these quinone derivatives indicated that the naphthoquinone ring and geranyl side-chain of geranylnaphthoquinone were respectively biosynthesized through the shikimate and methylerythritol phosphate pathways, and that these quinone derivatives have the same biosynthetic origin.     

Spermine and spermidine inhibition of photosystem II: Disassembly of the oxygen evolving complex and consequent perturbation in electron donation from TyrZ to P680 and the quinone acceptors QA to QB

Polyamines are implicated in plant growth and stress response. However, the polyamines spermine and spermidine were shown to elicit strong inhibitory effects in photosystem II (PSII) submembrane fractions. We have studied the mechanism of this inhibitory action in detail. The inhibition of electron transport in PSII submembrane fractions treated with millimolar concentrations of spermine or spermidine led to the decline of plastoquinone reduction, which was reversed by the artificial electron donor diphenylcarbazide. The above inhibition was due to the loss of the extrinsic polypeptides associated with the oxygen evolving complex. Thermoluminescence measurements revealed that charge recombination between the quinone acceptors of PSII, Q_A and Q_B, and the S₂ state of the Mn-cluster was abolished. Also, the dark decay of chlorophyll fluorescence after a single turn-over white flash was greatly retarded indicating a slower rate of Q_A⁻ reoxidation.     

Electron transfer patterns of the di-heme protein cytochrome c4 from Pseudomonas stutzeri

We report kinetic data for the two-step electron transfer (ET) oxidation and reduction of the two-domain di-heme redox protein Pseudomonas stutzeri cytochrome (cyt) c₄ by [Co(bipy)₃]^2+/3+ (bipy = 2,2′-bipyridine). Following earlier reports, the data accord with both bi- and tri-exponential kinetics. A complete kinetic scheme includes both “cooperative” intermolecular ET between each heme group and the external reaction partner, and intramolecular ET between the two heme groups. A new data analysis scheme shows unequivocally that two-ET oxidation and reduction of P. stutzeri cyt c₄ is entirely dominated by intermolecular ET between the heme groups and the external reaction partner in the ms time range, with virtually no contribution from intramolecular interheme ET in this time range. This is in striking contrast to two-ET electrochemical oxidation or reduction of P. stutzeri cyt c₄ for which fast, ms to sub-ms intramolecular interheme ET is a crucial step. The rate constant dependence on the solvent viscosity has disclosed strong coupling to both a (set of) frictionally damped solvent/protein nuclear modes and intramolecular friction-less “ballistic” modes, indicative of notable protein structural mobility in the overall two-ET process. We suggest that conformational protein mobility blocks intramolecular interheme ET in bulk homogeneous solution but triggers opening of this gated ET channel in the electrochemical environment or in the membrane environment of natural respiratory cyt c₄ function.     

Research of the (E/Z)-isomerization of carotenoids in Pécs since the 1970s

Geometrical configuration of the polyene chain of approximately 40 mono- and di-cis carotenoids was determined from 1970 through 1990. Subsequently, the kinetic, equilibrium and thermodynamic parameters (k, K, A, E_A, ΔH^#, ΔG^#, ΔS^#) of the reversible thermal isomerization of several symmetrical and unsymmetrical carotenoids were calculated. The rate of the iodine-catalyzed photoisomerization of (all-E)-, (9Z)- and (13Z)-zeaxanthin was compared and the ‘specific rate’ (per unit light energy at given wavelengths) of the iodine-catalyzed photoisomerization for several (13Z)-carotenoids was investigated. As the missing links of the biosynthetic pathway of paprika-carotenoids, carotenoids containing new end groups were isolated; their sterically unhindered mono-cis isomers were also prepared and their geometrical configuration was determined. The investigation concentrated on the substrate specificity of the enzyme violaxanthin-deepoxidase, the light-induced formation of (13Z)-violaxanthin in green leaves, the binding of xanthophylls to the bulk light-harvesting complex (LHC) of photosystem II in higher plants, the biochemical basis of color as an aesthetic quality in Citrus-fruits and the (9Z)-epoxycarotenoid cleavage enzyme activity for ABA biosynthesis. Recently (9Z)-capsanthin-5,6-epoxide and capsoneoxanthin, two novel carotenoids have been isolated from natural sources.     

Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in QB-site of Chlamydomonas reinhardtii

Tenuazonic acid (TeA) is a natural phytotoxin produced by Alternaria alternata, the causal agent of brown leaf spot disease of Eupatorium adenophorum. Results from chlorophyll fluorescence revealed TeA can block electron flow from Q_A to Q_B at photosystem II acceptor side. Based on studies with D1-mutants of Chlamydomonas reinhardtii, the No. 256 amino acid plays a key role in TeA binding to the Q_B-niche. The results of competitive replacement with [¹⁴C]atrazine combined with JIP-test and D1-mutant showed that TeA should be considered as a new type of photosystem II inhibitor because it has a different binding behavior within Q_B-niche from other known photosystem II inhibitors. Bioassay of TeA and its analogues indicated 3-acyl-5-alkyltetramic and even tetramic acid compounds may represent a new structural framework for photosynthetic inhibitors.