Internal charge transfer in cytochrome c oxidase at a limited proton supply: Proton pumping ceases at high pH

Background

In the membrane-bound enzyme cytochrome c oxidase, electron transfer from cytochrome c to O₂ is linked to proton uptake from solution to form H₂O, resulting in a charge separation across the membrane. In addition, the reaction drives pumping of protons across the membrane.

Methods

In this study we have measured voltage changes as a function of pH during reaction of the four-electron reduced cytochrome c oxidase from Rhodobacter sphaeroides with O₂. These electrogenic events were measured across membranes containing purified enzyme reconstituted into lipid vesicles.

Results

The results show that the pH dependence of voltage changes (primarily associated with proton transfer) during O₂ reduction does not match that of the previously studied absorbance changes (primarily associated with electron transfer). Furthermore, the voltage changes decrease with increasing pH.

Conclusions

The data indicate that cytochrome c oxidase does not pump protons at high pH (10.5) (or protons are taken from the “wrong” side of the membrane) and that at this pH the net proton-uptake stoichiometry is ∼ 1/2 of that at pH 8. Furthermore, the results provide a basis for interpretation of results from studies of mutant forms of the enzyme.

General significance

These results provide new insights into the function of cytochrome c oxidase.     

Mechanism and energetics by which glutamic acid 242 prevents leaks in cytochrome c oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme of aerobic respiration. The energy released from the reduction of molecular oxygen to water is used to pump protons across the mitochondrial or bacterial membrane. The pump function introduces a mechanistic requirement of a valve that prevents protons from flowing backwards during the process. It was recently found that Glu-242, a key amino acid in transferring protons to be pumped across the membrane and to the site of oxygen reduction, fulfils the function of such a valve by preventing simultaneous contact to the pump site and to the proton-conducting D-channel (Kaila V.R.I. et al. Proc. Natl. Acad. Sci. USA 105, 2008). Here we have incorporated the valve model into the framework of the reaction mechanism. The function of the Glu valve is studied by exploring how the redox state of the surrounding metal centers, dielectric effects, and membrane potential, affects the energetics and leaks of this valve. Parallels are drawn between the dynamics of Glu-242 and the long-standing obscure difference between the metastable O_H and stable O states of the binuclear center. Our model provides a suggestion for why reduction of the former state is coupled to proton translocation while reduction of the latter is not.     

Reaction of wild-type and Glu243Asp variant yeast cytochrome c oxidase with O2

We have studied internal electron transfer during the reaction of Saccharomyces cerevisiae mitochondrial cytochrome c oxidase with dioxygen. Similar absorbance changes were observed with this yeast oxidase as with the previously studied Rhodobacter sphaeroides and bovine mitochondrial oxidases, which suggests that the reaction proceeds along the same trajectory. However, notable differences were observed in rates and electron-transfer equilibrium constants of specific reaction steps, for example the ferryl (F) to oxidized (O) reaction was faster with the yeast (0.4 ms) than with the bovine oxidase (~ 1 ms) and a larger fraction Cu_A was oxidized with the yeast than with the bovine oxidase in the peroxy (P_R) to F reaction. Furthermore, upon replacement of Glu243, located at the end of the so-called D proton pathway, by Asp the P_R → F and F → O reactions were slowed by factors of ~ 3 and ~ 10, respectively, and electron transfer from Cu_A to heme a during the P_R → F reaction was not observed. These data indicate that during reduction of dioxygen protons are transferred through the D pathway, via Glu243, to the catalytic site in the yeast mitochondrial oxidase. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.     

Elevated Proton Leak of the Intermediate OH in Cytochrome c Oxidase

The kinetics of the formation and relaxation of transmembrane electric potential (Δψ) during the complete single turnover of CcO was studied in the bovine heart mitochondrial and the aa₃-type Paracoccus denitrificans enzymes incorporated into proteoliposome membrane. The real-time Δψ kinetics was followed by the direct electrometry technique. The prompt oxidation of CcO and formation of the activated, oxidized (O_H) state of the enzyme leaves the enzyme trapped in the open state that provides an internal leak for protons and thus facilitates dissipation of Δψ (τ^app ≤ 0.5-0.8 s). By contrast, when the enzyme in the O_H state is rapidly re-reduced by sequential electron delivery, Δψ dissipates much slower (τ^app > 3 s). In P. denitrificans CcO proteoliposomes the accelerated Δψ dissipation is slowed down by a mutational block of the proton conductance through the D-, but not K-channel. We concluded that in contrast to the other intermediates the O_H state of CcO is vulnerable to the elevated internal proton leak that proceeds via the D-channel.     

Transmembrane proton translocation by cytochrome c oxidase

Respiratory heme-copper oxidases are integral membrane proteins that catalyze the reduction of molecular oxygen to water using electrons donated by either quinol (quinol oxidases) or cytochrome c (cytochrome c oxidases, CcOs). Even though the X-ray crystal structures of several heme-copper oxidases and results from functional studies have provided significant insights into the mechanisms of O₂-reduction and, electron and proton transfer, the design of the proton-pumping machinery is not known. Here, we summarize the current knowledge on the identity of the structural elements involved in proton transfer in CcO. Furthermore, we discuss the order and timing of electron-transfer reactions in CcO during O₂ reduction and how these reactions might be energetically coupled to proton pumping across the membrane.     

Genetic incorporation of a photo-crosslinkable amino acid reveals novel protein complexes with GRB2 in mammalian cells

Cell signaling pathways are essentially organized through the distribution of various types of binding domains in signaling proteins, with each domain binding to specific target molecules. Although identification of these targets is crucial for mapping the pathways, affinity-based or copurification methods are insufficient to distinguish between direct and indirect interactions in a cellular context. In the present study, we developed another approach involving the genetic encoding of a photo-crosslinkable amino acid. p-Trifluoromethyl-diazirinyl-l-phenylalanine was thus incorporated at a defined site in the Src homology 2 (SH2) domain of the adaptor protein GRB2 in human embryonic kidney cells. These cells were exposed to 365-nm light after an epidermal growth factor stimulus, and the crosslinkable GRB2-SH2 domain exclusively formed covalent bonds with directly interacting proteins. Proteomic mass spectrometry analysis identified these direct binders of GRB2-SH2 separately from the proteins noncovalently bound to the Src homology 3 domains of GRB2. In addition to two signaling-associated proteins (GIT1 and AF6), the heterogeneous nuclear ribonucleoproteins F, H1, and H2 were thus identified as novel direct binders. The results revealed a connection between the cell signaling protein and the nuclear machinery involved in mRNA processing, and demonstrated the usefulness of genetically encoded photo-crosslinkers for mapping protein-protein interactions in cells.     

Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition

Background

DNA replication represents a critical step of the cell cycle which requires highly controlled and ordered regulatory mechanisms to ensure the integrity of genome duplication. Among a plethora of elements, post-translational modifications (PTMs) ensure the spatiotemporal regulation of pivotal proteins orchestrating cell division. Despite increasing evidences showing that O-GlcNAcylation regulates mitotic events, the impact of this PTM in the early steps of the cell cycle remains poorly understood.

Methods and results

Quiescent MCF7 cells were stimulated by serum mitogens and cell cycle progression was determined by flow cytometry. The levels of O-GlcNAc modified proteins, O-GlcNAc Transferase (OGT) and O-GlcNAcase (OGA) were examined by Western blotting and OGA activity was measured during the progression of cells towards S phase. A global decrease in O-GlcNAcylation was observed at S phase entry, concomitantly to an increase in the activity of OGA. A combination of two-dimensional electrophoresis, Western blotting and mass spectrometry was then used to detect and identify cell cycle-dependent putative O-GlcNAcylated proteins. 58 cytoplasmic and nuclear proteins differentially O-GlcNAcylated through G1/S transition were identified and the O-GlcNAc variations of Cytokeratin 8, hnRNP K, Caprin-1, Minichromosome Maintenance proteins MCM3, MCM6 and MCM7 were validated by immunoprecipitation.

Conclusions

The dynamics of O-GlcNAc is regulated during G1/S transition and observed on key proteins involved in the cytoskeleton networks, mRNA processing, translation, protein folding and DNA replication.

General significance

Our results led us to propose that O-GlcNAcylation joins the PTMs that take part in the regulation of DNA replication initiation.     

Allelopathy is involved in the formation of pure colonies of the fern Gleichenia japonica

The fern Gleichenia japonica is one of the most widely distributed fern and occurs throughout East to South Asia. The species often dominates plant communities by forming large monospecific colonies. However, the potential mechanism for this domination has not yet been described. The objective of this study was to test the hypothesis that allelochemicals are involved in the formation of G. japonica colonies. An aqueous methanol extract of G. japonica inhibited the growth of seedlings of garden cress (Lepidium sativum), lettuce (Lactuca sativa), ryegrass (Lolium multiflorum) and timothy (Phleum pratense). Increasing extract concentration increased the inhibition. These results suggest that G. japonica contain allelopathic substances. The extract was then purified by several chromatographies with monitoring the inhibitory activity and two growth inhibitory substances causing the allelopathic effect were isolated. The chemical structures of the two substances were determined by spectral data to be a novel compound 3-O-β-allopyranosyl-13-O-β-fucopyranosyl-3β-hydroxymanool (1) and 18-O-α-l-rhamnopyranosyl-(1→2)-β-d-glucopyranosyl-13-epitorreferol (2). These compounds inhibited the shoot and root growth of garden cress, lettuce, alfalfa (Medicago sativa), timothy, ryegrass and barnyardgrass (Echinochloa crus-galli) at concentrations greater than 0.1–1.0 mM. The concentrations required for 50% growth inhibition of root and shoot growth of these test plants ranged from 0.72 to 3.49 mM and 0.79 to 3.51 mM for compounds 1 and 2, respectively. Concentration of compounds 1 and 2 in soil under the pure colony of G. japonica was 4.9 and 5.7 mM, respectively, indicating concentrations over those required for 50% growth inhibition are potentially available under monocultural stands of these ferns. Therefore, these compounds may contribute to the allelopathic effects caused by presence of G. japonica and may thus contribute to the establishment of monocultural stands by this fern.     

The high turnover Drosophila multidrug resistance-associated protein shares the biochemical features of its human orthologues

DMRP, an ABC transporter encoded by the dMRP/CG6214 gene, is the Drosophila melanogaster orthologue of the “long” human multidrug resistance-associated proteins (MRP1/ABCC1, MRP2/ABCC2, MRP3/ABCC3, MRP6/ABCC6, and MRP7/ABCC10). In order to provide a detailed biochemical characterisation we expressed DMRP in Sf9 insect cell membranes. We demonstrated DMRP as a functional orthologue of its human counterparts capable of transporting several human MRP substrates like β-estradiol 17-β-d-glucuronide, leukotriene C₄, calcein, fluo3 and carboxydichlorofluorescein. Unexpectedly, we found DMRP to exhibit an extremely high turnover rate for the substrate transport as compared to its human orthologues. Furthermore, DMRP showed remarkably high basal ATPase activity (68-75 nmol P_i/mg membrane protein/min), which could be further stimulated by probenecid and the glutathione conjugate of N-ethylmaleimide. Surprisingly, this high level basal ATPase activity was inhibited by the transported substrates. We discussed this phenomenon in the light of a potential endogenous substrate (or activator) present in the Sf9 membrane.     

Insights into the function of PsbR protein in Arabidopsis thaliana

The functional state of the Photosystem (PS) II complex in Arabidopsis psbR T-DNA insertion mutant was studied. The ΔPsbR thylakoids showed about 34% less oxygen evolution than WT, which correlates with the amounts of PSII estimated from Y_D^ox radical EPR signal. The increased time constant of the slow phase of flash fluorescence (FF)-relaxation and upshift in the peak position of the main TL-bands, both in the presence and in the absence of DCMU, confirmed that the S₂Q_A⁻ and S₂Q_B⁻ charge recombinations were stabilized in ΔPsbR thylakoids. Furthermore, the higher amount of dark oxidized Cyt-b559 and the increased proportion of fluorescence, which did not decay during the 100s time span of the measurement thus indicating higher amount of Y_D⁺Q_A⁻ recombination, pointed to the donor side modifications in ΔPsbR. EPR measurements revealed that S₁-to-S₂-transition and S₂-state multiline signal were not affected by mutation. The fast phase of the FF-relaxation in the absence of DCMU was significantly slowed down with concomitant decrease in the relative amplitude of this phase, indicating a modification in Q_A to Q_B electron transfer in ΔPsbR thylakoids. It is concluded that the lack of the PsbR protein modifies both the donor and the acceptor side of the PSII complex.     

ABC transporters do not contribute to extracellular translocation of hyaluronan in human breast cancer in vitro

Extracellular translocation of the polysaccharide, hyaluronan (HA) has been thought to be mediated via its transmembrane synthetic enzyme, hyaluronan synthase (HAS) but recent studies have indicated that the ATP-Binding-Cassette (ABC) transporter, MRP5 contributes to this process. Liberated and cell-associated HA contributes to breast cancer initiation and progression, and therefore the inhibition of ABC transporters and consequently HA transport could provide therapeutic benefit in the treatment of breast cancer. Quantitation of ABC transporter genes, MRP1-5, BCRP and MDR1 were determined in six breast cancer cell lines selected for their differential HA synthetic rates. Low endogenous expression of transporters was detected but no significant correlation existed between ABC transporter and HAS gene expression or HA production. A dose titration of up to ten times the IC₅₀ of ten small molecule ABC transporter inhibitors did not significantly inhibit HA export in four breast cancer cell lines. Unlike the changes observed after inhibition of HA synthesis by the characterised inhibitor 4-MU, inhibition of ABC transporters did not alter the cell morphology, HA glycocalyx or the intracellular quantity or localisation of HA. Collectively these data indicate that ABC transporters do not contribute to the extracellular transport of HA in breast cancer, supporting a role for the hyaluronan synthase in translocation.     

SSO1450 - A CAS1 protein from Sulfolobus solfataricus P2 with high affinity for RNA and DNA

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are ubiquitous in archaea and eubacteria. It has been suggested that CRISPR and CAS proteins act as an immune system preventing the invasion of foreign genomic elements at the DNA level. The protein SSO1450 from Sulfolobus solfataricus (Sso) P2 belongs to the CAS1 cluster which is one of the core protein clusters most frequently associated with CRISPR sequences. In this study we show that SSO1450 is a high-affinity nucleic acid binding protein. It binds DNA, RNA and DNA-RNA hybrid apparently sequence non-specific in a multi-site binding mode. Furthermore, SSO1450 promotes the hybridization of complementary nucleic acid strands.     

Hydrogen-bond formation of the residue in H-loop of the nucleotide binding domain 2 with the ATP in this site and/or other residues of multidrug resistance protein MRP1 plays a crucial role during ATP-dependent solute transport

MRP1 couples ATP binding/hydrolysis to solute transport. We have shown that ATP binding to nucleotide-binding-domain 1 (NBD1) plays a regulatory role whereas ATP hydrolysis at NBD2 plays a crucial role in ATP-dependent solute transport. However, how ATP is hydrolyzed at NBD2 is not well elucidated. To partially address this question, we have mutated the histidine residue in H-loop of MRP1 to either a residue that prevents the formation of hydrogen-bonds with ATP and other residues in MRP1 or a residue that may potentially form these hydrogen-bonds. Interestingly, substitution of H827 in NBD1 with residues that prevented formation of these hydrogen-bonds had no effect on the ATP-dependent solute transport whereas corresponding mutations in NBD2 almost abolished the ATP-dependent solute transport completely. In contrast, substitutions of H1486 in H-loop of NBD2 with residues that might potentially form these hydrogen-bonds exerted either full function or partial function, implying that hydrogen-bond formation between the residue at 1486 and the γ-phosphate of the bound ATP and/or other residues, such as putative catalytic base E1455, together with S769, G771, T1329 and K1333, etc., holds all the components necessary for ATP binding/hydrolysis firmly so that the activated water molecule can efficiently hydrolyze the bound ATP at NBD2.     

Effects of precipitation on photosynthesis and water potential in Andropogon gerardii and Schizachyrium scoparium in a southern mixed grass prairie

In grassland ecosystems, spatial and temporal variability in precipitation is a key driver of species distributions and population dynamics. We experimentally manipulated precipitation to understand the physiological basis for differences in responses of species to water availability in a southern mixed grass prairie. We focused on the performance of two dominant C₄ grasses, Andropogon gerardii Vitman and Schizachyrium scoparium (Michx.) Nash, in treatments that received ambient rainfall, half of ambient rainfall (“drought” treatment), or approximately double ambient rainfall (“irrigated” treatment). Water potentials of S. scoparium were lower than A. gerardii, suggesting superior ability to adjust to water deficit in S. scoparium. Additionally, drought reduced photosynthesis to a greater extent in A. gerardii compared to S. scoparium. Leaf-level photosynthesis rates were similar in ambient and irrigated treatments, but were significantly lower in the drought treatment. Although stomatal conductance was reduced by drought, this was not limiting for photosynthesis. Leaf δ¹³C values were decreased by drought, caused by an increase in C_i/C_a. Chlorophyll fluorescence measures indicated light-harvesting rates were highest in irrigated treatments, and were lower in ambient and drought treatments. Moreover, drought resulted in a greater proportion of absorbed photon energy being lost via thermal pathways. Reductions in photosynthesis came as a result of non-stomatal limitations in the C₄ cycle. Our results provide mechanistic support for the hypothesis that S. scoparium is more drought tolerant than A. gerardii.     

Unusual thermal stability of RNA/[RP-PS]-DNA/RNA triplexes containing a homopurine DNA strand

Homopurine deoxyribonucleoside phosphorothioates, as short as hexanucleotides and possessing all internucleotide linkages of RP configuration, form a triple helix with two RNA or 2'-OMe-RNA strands, with Watson-Crick and Hoogsteen complementarity. Melting temperature and fluorescence quenching experiments strongly suggest that the Hoogsteen RNA strand is parallel to the homopurine [RP-PS]-oligomer. Remarkably, these triplexes are thermally more stable than complexes formed by unmodified homopurine DNA molecules of the same sequence. The triplexes formed by phosphorothioate DNA dodecamers containing 4-6 dG residues are thermally stable at pH 7.4, although their stability increases significantly at pH 5.3. FTIR measurements suggest participation of the C2-carbonyl group of the pyrimidines in the stabilization of the triplex structure. Formation of triple-helix complexes with exogenously delivered PS-oligos may become useful for the reduction of RNA accessibility in vivo and, hence, selective suppression/inhibition of the translation process.