Identification of the proton pathway in bacterial reaction centers: Inhibition of proton transfer by binding of Zn2+ or Cd2+

The reaction center (RC) from Rhodobacter sphaeroides converts light into chemical energy through the light induced two-electron, two-proton reduction of a bound quinone molecule QB (the secondary quinone acceptor). A unique pathway for proton transfer to the QB site had so far not been determined. To study the molecular basis for proton transfer, we investigated the effects of exogenous metal ion binding on the kinetics of the proton-assisted electron transfer kAB(2) (QA−•QB−• + H+ → QA(QBH)−, where QA is the primary quinone acceptor). Zn2+ and Cd2+ bound stoichiometrically to the RC (KD ≤ 0.5 μM) and reduced the observed value of kAB(2) 10-fold and 20-fold (pH 8.0), respectively. The bound metal changed the mechanism of the kAB(2) reaction. In native RCs, kAB(2) was previously shown to be rate-limited by electron transfer based on the dependence of kAB(2) on the driving force for electron transfer. Upon addition of Zn2+ or Cd2+, kAB(2) became approximately independent of the electron driving force, implying that the rate of proton transfer was reduced (≥ 102-fold) and has become the rate-limiting step. The lack of an effect of the metal binding on the charge recombination reaction D+•QAQB−• → DQAQB suggests that the binding site is located far (>10 Å) from QB. This hypothesis is confirmed by preliminary x-ray structure analysis. The large change in the rate of proton transfer caused by the stoichiometric binding of the metal ion shows that there is one dominant site of proton entry into the RC from which proton transfer to QB−• occurs.

Identification of the proton pathway in bacterial reaction centers: Both protons associated with reduction of QB to QBH2 share a common entry point

The reaction center from Rhodobacter sphaeroides uses light energy for the reduction and protonation of a quinone molecule, QB. This process involves the transfer of two protons from the aqueous solution to the protein-bound QB molecule. The second proton, H+(2), is supplied to QB by Glu-L212, an internal residue protonated in response to formation of QA− and QB−. In this work, the pathway for H+(2) to Glu-L212 was studied by measuring the effects of divalent metal ion binding on the protonation of Glu-L212, which was assayed by two types of processes. One was proton uptake from solution after the one-electron reduction of QA (DQA→D+QA−) and QB (DQB→D+QB−), studied by using pH-sensitive dyes. The other was the electron transfer kAB(1) (QA−QB→QAQB−). At pH 8.5, binding of Zn2+, Cd2+, or Ni2+ reduced the rates of proton uptake upon QA− and QB− formation as well as kAB(1) by ≈an order of magnitude, resulting in similar final values, indicating that there is a common rate-limiting step. Because D+QA− is formed 105-fold faster than the induced proton uptake, the observed rate decrease must be caused by an inhibition of the proton transfer. The Glu-L212→Gln mutant reaction centers displayed greatly reduced amplitudes of proton uptake and exhibited no changes in rates of proton uptake or electron transfer upon Zn2+ binding. Therefore, metal binding specifically decreased the rate of proton transfer to Glu-L212, because the observed rates were decreased only when proton uptake by Glu-L212 was required. The entry point for the second proton H+(2) was thus identified to be the same as for the first proton H+(1), close to the metal binding region Asp-H124, His-H126, and His-H128.

Photochemically induced nuclear spin polarization in reaction centers of photosystem II observed by 13C-solid-state NMR reveals a strongly asymmetric electronic structure of the P680.+ primary donor chlorophyll

We report 13C magic angle spinning NMR observation of photochemically induced dynamic nuclear spin polarization (photo- CIDNP) in the reaction center (RC) of photosystem II (PS2). The light-enhanced NMR signals of the natural abundance 13C provide information on the electronic structure of the primary electron donor P680 (chlorophyll a molecules absorbing around 680 nm) and on the pz spin density pattern in its oxidized form, P680⨥. Most centerband signals can be attributed to a single chlorophyll a (Chl a) cofactor that has little interaction with other pigments. The chemical shift anisotropy of the most intense signals is characteristic for aromatic carbon atoms. The data reveal a pronounced asymmetry of the electronic spin density distribution within the P680⨥. PS2 shows only a single broad and intense emissive signal, which is assigned to both the C-10 and C-15 methine carbon atoms. The spin density appears shifted toward ring III. This shift is remarkable, because, for monomeric Chl a radical cations in solution, the region of highest spin density is around ring II. It leads to a first hypothesis as to how the planet can provide itself with the chemical potential to split water and generate an oxygen atmosphere using the Chl a macroaromatic cycle. A local electrostatic field close to ring III can polarize the electronic charge and associated spin density and increase the redox potential of P680 by stabilizing the highest occupied molecular orbital, without a major change of color. This field could be produced, e.g., by protonation of the keto group of ring V. Finally, the radical cation electronic structure in PS2 is different from that in the bacterial RC, which shows at least four emissive centerbands, indicating a symmetric spin density distribution over the entire bacteriochlorophyll macrocycle.

Hydrolysis of GTP on rab11 is required for the direct delivery of transferrin from the pericentriolar recycling compartment to the cell surface but not from sorting endosomes

Rab11 is a small GTP-binding protein that in cultured mammalian cells has been shown to be concentrated in the pericentriolar endosomal recycling compartment and to play a key role in passage of the recycling transferrin receptor through that compartment [Ullrich, O., Reinsch, S., Urbé, S., Zerial, M. & Parton, R. G. (1996) J. Cell Biol. 135, 913–924]. To obtain insights into the site(s) of action of rab11 within the recycling pathway, we have now compared the effects on recycling at 37°C of overexpression of wild-type rab11 and various mutant forms of this protein in cells that had been loaded with transferrin at either 37°C or 16°C. We show that incubation at 16°C blocks passage of endocytosed transferrin into the recycling compartment and that, whereas the rab11 dominant negative mutant form (S25N) inhibits transferrin recycling after interiorization at either temperature, the wild-type rab11 and constitutively active mutant (Q70L) have no inhibitory effect on the recycling of molecules that were interiorized at 16°C. This differential inhibitory effect shows that two distinct pathways for recycling are followed by the bulk of the transferrin molecules interiorized at the two different temperatures. The incapacity of the constitutively active form of rab11 (Q70L) to inhibit recycling of molecules interiorized at 16°C is consistent with their recycling taking place directly from sorting endosomes, in a process that does not require hydrolysis of GTP on rab11. The fact that the dominant negative (S25N) form of rab11 inhibits recycling of molecules interiorized at both temperatures indicates that activation of rab11 by GTP is required for exit of transferrin from sorting endosomes, regardless of whether this exit is toward the recycling compartment or directly to the plasma membrane.

Relationship between the oxidation potential and electron spin density of the primary electron donor in reaction centers from Rhodobacter sphaeroides

The primary electron donor in bacterial reaction centers is a dimer of bacteriochlorophyll a molecules, labeled L or M based on their proximity to the symmetry-related protein subunits. The electronic structure of the bacteriochlorophyll dimer was probed by introducing small systematic variations in the bacteriochlorophyll–protein interactions by a series of site-directed mutations that replaced residue Leu M160 with histidine, tyrosine, glutamic acid, glutamine, aspartic acid, asparagine, lysine, and serine. The midpoint potentials for oxidation of the dimer in the mutants showed an almost continuous increase up to ≈60 mV compared with wild type. The spin density distribution of the unpaired electron in the cation radical state of the dimer was determined by electron–nuclear–nuclear triple resonance spectroscopy in solution. The ratio of the spin density on the L side of the dimer to the M side varied from ≈2:1 to ≈5:1 in the mutants compared with ≈2:1 for wild type. The correlation between the midpoint potential and spin density distribution was described using a simple molecular orbital model, in which the major effect of the mutations is assumed to be a change in the energy of the M half of the dimer, providing estimates for the coupling and energy levels of the orbitals in the dimer. These results demonstrate that the midpoint potential can be fine-tuned by electrostatic interactions with amino acids near the dimer and show that the properties of the electronic structure of a donor or acceptor in a protein complex can be directly related to functional properties such as the oxidation–reduction midpoint potential.

Ascorbate recycling in human neutrophils: Induction by bacteria

Ascorbate (vitamin C) recycling occurs when extracellular ascorbate is oxidized, transported as dehydroascorbic acid, and reduced intracellularly to ascorbate. We investigated microorganism induction of ascorbate recycling in human neutrophils and in microorganisms themselves. Ascorbate recycling was determined by measuring intracellular ascorbate accumulation. Ascorbate recycling in neutrophils was induced by both Gram-positive and Gram-negative pathogenic bacteria, and the fungal pathogen Candida albicans. Induction of recycling resulted in as high as a 30-fold increase in intracellular ascorbate compared with neutrophils not exposed to microorganisms. Recycling occurred at physiologic concentrations of extracellular ascorbate within 20 min, occurred over a 100-fold range of effector/target ratios, and depended on oxidation of extracellular ascorbate to dehydroascorbic acid. Ascorbate recycling did not occur in bacteria nor in C. albicans. Ascorbate did not enter microorganisms, and dehydroascorbic acid entry was less than could be accounted for by diffusion. Because microorganism lysates reduced dehydroascorbic acid to ascorbate, ascorbate recycling was absent because of negligible entry of the substrate dehydroascorbic acid. Because ascorbate recycling occurs in human neutrophils but not in microorganisms, it may represent a eukaryotic defense mechanism against oxidants with possible clinical implications.

A Di-Leucine Sequence and a Cluster of Acidic Amino Acids Are Required for Dynamic Retention in the Endosomal Recycling Compartment of Fibroblasts

Insulin-regulated aminopeptidase (IRAP), a transmembrane aminopeptidase, is dynamically retained within the endosomal compartment of fibroblasts. The characteristics of this dynamic retention are rapid internalization from the plasma membrane and slow recycling back to the cell surface. These specialized trafficking kinetics result in <15% of IRAP on the cell surface at steady state, compared with 35% of the transferrin receptor, another transmembrane protein that traffics between endosomes and the cell surface. Here we demonstrate that a 29-amino acid region of IRAP's cytoplasmic domain (residues 56–84) is necessary and sufficient to promote trafficking characteristic of IRAP. A di-leucine sequence and a cluster of acidic amino acids within this region are essential elements of the motif that slows IRAP recycling. Rapid internalization requires any two of three distinct motifs: M15,16, DED64–66, and LL76,77. The DED and LL sequences are part of the motif that regulates recycling, demonstrating that this motif is bifunctional. In this study we used horseradish peroxidase quenching of fluorescence to demonstrate that IRAP is dynamically retained within the transferrin receptor-containing general endosomal recycling compartment. Therefore, our data demonstrate that motifs similar to those that determine targeting among distinct membrane compartments can also regulate the rate of transport of proteins from endosomal compartments. We propose a model for dynamic retention in which IRAP is transported from the general endosomal recycling compartment in specialized, slowly budding recycling vesicles that are distinct from those that mediate rapid recycling back to the surface (e.g., transferrin receptor-containing transport vesicles). It is likely that the dynamic retention of IRAP is an example of a general mechanism for regulating the distribution of proteins between the surface and interior of cells.

Identification of Discrete Classes of Endosome-derived Small Vesicles as a Major Cellular Pool for Recycling Membrane Proteins

Vesicles carrying recycling plasma membrane proteins from early endosomes have not yet been characterized. Using Chinese hamster ovary cells transfected with the facilitative glucose transporter, GLUT4, we identified two classes of discrete, yet similarly sized, small vesicles that are derived from early endosomes. We refer to these postendosomal vesicles as endocytic small vesicles or ESVs. One class of ESVs contains a sizable fraction of the pool of the transferrin receptor, and the other contains 40% of the total cellular pool of GLUT4 and is enriched in the insulin-responsive aminopeptidase (IRAP). The ESVs contain cellubrevin and Rab4 but are lacking other early endosomal markers, such as EEA1 or syntaxin13. The ATP-, temperature-, and cytosol-dependent formation of ESVs has been reconstituted in vitro from endosomal membranes. Guanosine 5′-[γ-thio]triphosphate and neomycin, but not brefeldin A, inhibit budding of the ESVs in vitro. A monoclonal antibody recognizing the GLUT4 cytoplasmic tail perturbs the in vitro targeting of GLUT4 to the ESVs without interfering with the incorporation of IRAP or TfR. We suggest that cytosolic proteins mediate the incorporation of recycling membrane proteins into discrete populations of ESVs that serve as carrier vesicles to store and then transport the cargo from early endosomes, either directly or indirectly, to the cell surface.

The Human Cytomegalovirus US28 Protein Is Located in Endocytic Vesicles and Undergoes Constitutive Endocytosis and Recycling

Genes encoding chemokine receptor-like proteins have been found in herpes and poxviruses and implicated in viral pathogenesis. Here we describe the cellular distribution and trafficking of a human cytomegalovirus (HCMV) chemokine receptor encoded by the US28 gene, after transient and stable expression in transfected HeLa and Cos cells. Immunofluorescence staining indicated that this viral protein accumulated intracellularly in vesicular structures in the perinuclear region of the cell and showed overlap with markers for endocytic organelles. By immunogold electron microscopy US28 was seen mostly to localize to multivesicular endosomes. A minor portion of the protein (at most 20%) was also expressed at the cell surface. Antibody-feeding experiments indicated that cell surface US28 undergoes constitutive ligand-independent endocytosis. Biochemical analysis with the use of iodinated ligands showed that US28 was rapidly internalized. The high-affinity ligand of US28, the CX3C-chemokine fractalkine, reduced the steady-state levels of US28 at the cell surface, apparently by inhibiting the recycling of internalized receptor. Endocytosis and cycling of HCMV US28 could play a role in the sequestration of host chemokines, thereby modulating antiviral immune responses. In addition, the distribution of US28 mainly on endosomal membranes may allow it to be incorporated into the viral envelope during HCMV assembly.

Myosin Vb Is Associated with Plasma Membrane Recycling Systems

Myosin Va is associated with discrete vesicle populations in a number of cell types, but little is known of the function of myosin Vb. Yeast two-hybrid screening of a rabbit parietal cell cDNA library with dominant active Rab11a (Rab11aS20V) identified myosin Vb as an interacting protein for Rab11a, a marker for plasma membrane recycling systems. The isolated clone, corresponding to the carboxyl terminal 60 kDa of the myosin Vb tail, interacted with all members of the Rab11 family (Rab11a, Rab11b, and Rab25). GFP-myosin Vb and endogenous myosin Vb immunoreactivity codistributed with Rab11a in HeLa and Madin-Darby canine kidney (MDCK) cells. As with Rab11a in MDCK cells, the myosin Vb immunoreactivity was dispersed with nocodazole treatment and relocated to the apical corners of cells with taxol treatment. A green fluorescent protein (GFP)-myosin Vb tail chimera overexpressed in HeLa cells retarded transferrin recycling and caused accumulation of transferrin and the transferrin receptor in pericentrosomal vesicles. Expression of the myosin Vb tail chimera in polarized MDCK cells stably expressing the polymeric IgA receptor caused accumulation of basolaterally endocytosed polymeric IgA and the polymeric IgA receptor in the pericentrosomal region. The myosin Vb tail had no effects on transferrin trafficking in polarized MDCK cells. The GFP-myosin Va tail did not colocalize with Rab11a and had no effects on recycling system vesicle distribution in either HeLa or MDCK cells. The results indicate myosin Vb is associated with the plasma membrane recycling system in nonpolarized cells and the apical recycling system in polarized cells. The dominant negative effects of the myosin Vb tail chimera indicate that this unconventional myosin is required for transit out of plasma membrane recycling systems.

Rad52 forms DNA repair and recombination centers during S phase

Maintenance of genomic integrity and stable transmission of genetic information depend on a number of DNA repair processes. Failure to faithfully perform these processes can result in genetic alterations and subsequent development of cancer and other genetic diseases. In the eukaryote Saccharomyces cerevisiae, homologous recombination is the major pathway for repairing DNA double-strand breaks. The key role played by Rad52 in this pathway has been attributed to its ability to seek out and mediate annealing of homologous DNA strands. In this study, we find that S. cerevisiae Rad52 fused to green fluorescent protein (GFP) is fully functional in DNA repair and recombination. After induction of DNA double-strand breaks by γ-irradiation, meiosis, or the HO endonuclease, Rad52-GFP relocalizes from a diffuse nuclear distribution to distinct foci. Interestingly, Rad52 foci are formed almost exclusively during the S phase of mitotic cells, consistent with coordination between recombinational repair and DNA replication. This notion is further strengthened by the dramatic increase in the frequency of Rad52 focus formation observed in a pol12-100 replication mutant and a mec1 DNA damage checkpoint mutant. Furthermore, our data indicate that each Rad52 focus represents a center of recombinational repair capable of processing multiple DNA lesions.

Glutathione-mediated destabilization in vitro of [2Fe-2S] centers in the SoxR regulatory protein.

SoxR is a transcription factor that governs a global defense against the oxidative stress caused by nitric oxide or excess superoxide in Escherichia coli. SoxR is a homodimer containing a pair of [2Fe-2S] clusters essential for its transcriptional activity, and changes in the stability of these metal centers could contribute to the activation or inactivation of SoxR in vivo. Herein we show that reduced glutathione (GSH) in aerobic solution disrupts the SoxR [2Fe-2S] clusters, releasing Fe from the protein and eliminating SoxR transcriptional activity. This disassembly process evidently involves oxygen-derived free radicals. The loss of [2Fe-2S] clusters does not occur in anaerobic solution and is blocked in aerobic solution by the addition of superoxide dismutase and catalase. Although H2O2 or xanthine oxidase and hypoxanthine (to generate superoxide) were insufficient on their own to cause [2Fe-2S] cluster loss, they did accelerate the rate of disassembly after GSH addition. Oxidized GSH alone was ineffective in disrupting the clusters, but the rate of [2Fe-2S] cluster disassembly was maximal when reduced and oxidized GSH were present at a ratio of approximately 1:3, which suggests the critical involvement of a GSH-based free radical in the disassembly process. Such a reaction might occur in vivo: we found that the induction by paraquat of SoxR-dependent soxS transcription was much higher in a GSH-deficient E. coli strain than in its GSH-containing parent. The results imply that GSH may play a significant role during the deactivation process of SoxR in vivo. Ironically, superoxide production seems both to activate SoxR and, in the GSH-dependent disassembly process, to switch off this transcription factor.

Rab4 and cellubrevin define different early endosome populations on the pathway of transferrin receptor recycling.

During receptor mediated endocytosis, at least a fraction of recycling cargo typically accumulates in a pericentriolar cluster of tubules and vesicles. However, it is not clear if these endosomal structures are biochemically distinct from the early endosomes from which they are derived. To better characterize this pericentriolar endosome population, we determined the distribution of two endogenous proteins known to be functionally involved in receptor recycling [Rab4, cellubrevin (Cbvn)] relative to the distribution of a recycling ligand [transferrin (Tfn)] as it traversed the endocytic pathway. Shortly after internalization, Tfn entered a population of early endosomes that contained both Rab4 and Cbvn, demonstrated by triple label immunofluorescence confocal microscopy. Tfn then accumulated in the pericentriolar cluster of recycling vesicles (RVs). However, although these pericentriolar endosomes contained Cbvn, they were strikingly depleted of Rab4. The ability of internalized Tfn to reach the Rab4-negative population was not blocked by nocodazole, although the characteristic pericentriolar location of the population was not maintained in the absence of microtubules. Similarly, Rab4-positive and -negative populations remained distinct in cells treated with brefeldin A, with only Rab4-positive elements exhibiting the extended tubular morphology induced by the drug. Thus, at least with respect to Rab4 distribution, the pathway of Tfn receptor recycling consists of at least two biochemically and functionally distinct populations of endosomes, a Rab4-positive population of early endosomes to which incoming Tfn is initially delivered and a Rab4-negative population of recycling vesicles that transiently accumulates Tfn on its route back to the plasma membrane.

Natural abundance solid-state carbon NMR studies of photosynthetic reaction centers with photoinduced polarization.

Solid-state NMR spectra of natural abundance 13C in reaction centers from photosynthetic bacteria Rhodobacter sphaeroides R-26 was measured. When the quinone acceptors were removed and continuous visible illumination of the sample was provided, exceptionally strong nuclear spin polarization was observed in NMR lines with chemical shifts resembling those of the aromatic carbons in bacteriochlorophyll and bacteriopheophytin. The observation of spin polarized 15N nuclei in bacteriochlorophyll and bacteriopheophytin was previously demonstrated with nonspecifically 15N-labeled reaction centers. Both the carbon and the nitrogen NMR studies indicate that the polarization is developed on species that carry unpaired electrons in the early electron transfer steps, including the bacteriochlorophyll dimer donor P860 and probably the bacteriopheophytin acceptor. I. Both enhanced-absorptive and emissive polarization were seen in the carbon spectrum; most lines were absorptive but the methine carbons of the porphyrin ring (alpha, beta, gamma, ) exhibited emissive polarization. The change in the sign of the hyperfine coupling at these sites indicates the existence of nodes in the spin density distribution on the tetrapyrrole cofactors flanking each methine carbon bridge.

Aromatic amino acid transamination and methionine recycling in trypanosomatids.

Although trypanosomatids are known to rapidly transaminate exogenous aromatic amino acids in vitro and in vivo, the physiological significance of this reaction is not understood. In postmitochondrial supernatants prepared from Trypanosoma brucei brucei and Crithidia fasciculata, we have found that aromatic amino acids were the preferred amino donors for the transamination of alpha-ketomethiobutyrate to methionine. Intact C. fasciculata grown in the presence of [15N]tyrosine were found to contain detectable [15N]methionine, demonstrating that this reaction occurs in situ in viable cells. This process is the final step in the recycling of methionine from methylthioadenosine, a product of decarboxylated S-adenosylmethionine from the polyamine synthetic pathway. Mammalian liver, in contrast, preferentially used glutamine for this reaction and utilized a narrower range of amino donors than seen with the trypanosomatids. Studies with methylthioadenosine showed that this compound was readily converted to methionine, demonstrating a fully functional methionine-recycling pathway in trypanosomatids.