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1
Broo, Anders, and Sven Larsson. "Calculation of electron transfer rates in proteins." International Journal of Quantum Chemistry 36, S16 (June 19, 2009): 185–98. http://dx.doi.org/10.1002/qua.560360714.
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Gray, Harry B., and Jay R. Winkler. "Electron tunneling through proteins." Quarterly Reviews of Biophysics 36, no. 3 (August 2003): 341–72. http://dx.doi.org/10.1017/s0033583503003913.
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1. History 3422. Activation barriers 3432.1 Redox potentials 3442.2 Reorganization energy 3443. Electronic coupling 3454. Ru-modified proteins 3484.1 Reorganization energy 3494.1.1 Cyt c 3494.1.2 Azurin 3504.2 Tunneling timetables 3525. Multistep tunneling 3576. Protein–protein reactions 3596.1 Hemoglobin (Hb) hybrids 3596.2 Cyt c/cyt b5 complexes 3606.3 Cyt c/cyt c peroxidase complexes 3606.4 Zn–cyt c/Fe–cyt c crystals 3617. Photosynthesis and respiration 3627.1 Photosynthetic reaction centers (PRCs) 3627.2 Cyt c oxidase (CcO) 3648. Concluding remarks 3659. Acknowledgments 36610. References 366Electron transfer processes are vital elements of energy transduction pathways in living cells. More than a half century of research has produced a remarkably detailed understanding of the factors that regulate these ‘currents of life’. We review investigations of Ru-modified proteins that have delineated the distance- and driving-force dependences of intra-protein electron-transfer rates. We also discuss electron transfer across protein–protein interfaces that has been probed both in solution and in structurally characterized crystals. It is now clear that electrons tunnel between sites in biological redox chains, and that protein structures tune thermodynamic properties and electronic coupling interactions to facilitate these reactions. Our work has produced an experimentally validated timetable for electron tunneling across specified distances in proteins. Many electron tunneling rates in cytochrome c oxidase and photosynthetic reaction centers agree well with timetable predictions, indicating that the natural reactions are highly optimized, both in terms of thermodynamics and electronic coupling. The rates of some reactions, however, significantly exceed timetable predictions; it is likely that multistep tunneling is responsible for these anomalously rapid charge transfer events.
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Catarino, Teresa, and David L. Turner. "Thermodynamic Control of Electron Transfer Rates in Multicentre Redox Proteins." ChemBioChem 2, no. 6 (June 1, 2001): 416–24. http://dx.doi.org/10.1002/1439-7633(20010601)2:6<416::aid-cbic416>3.0.co;2-z.
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van Wonderen, Jessica H., Katrin Adamczyk, Xiaojing Wu, Xiuyun Jiang, Samuel E. H. Piper, Christopher R. Hall, Marcus J. Edwards, et al. "Nanosecond heme-to-heme electron transfer rates in a multiheme cytochrome nanowire reported by a spectrally unique His/Met-ligated heme." Proceedings of the National Academy of Sciences 118, no. 39 (September 23, 2021): e2107939118. http://dx.doi.org/10.1073/pnas.2107939118.
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Proteins achieve efficient energy storage and conversion through electron transfer along a series of redox cofactors. Multiheme cytochromes are notable examples. These proteins transfer electrons over distance scales of several nanometers to >10 μm and in so doing they couple cellular metabolism with extracellular redox partners including electrodes. Here, we report pump-probe spectroscopy that provides a direct measure of the intrinsic rates of heme–heme electron transfer in this fascinating class of proteins. Our study took advantage of a spectrally unique His/Met-ligated heme introduced at a defined site within the decaheme extracellular MtrC protein of Shewanella oneidensis. We observed rates of heme-to-heme electron transfer on the order of 109 s−1 (3.7 to 4.3 Å edge-to-edge distance), in good agreement with predictions based on density functional and molecular dynamics calculations. These rates are among the highest reported for ground-state electron transfer in biology. Yet, some fall 2 to 3 orders of magnitude below the Moser–Dutton ruler because electron transfer at these short distances is through space and therefore associated with a higher tunneling barrier than the through-protein tunneling scenario that is usual at longer distances. Moreover, we show that the His/Met-ligated heme creates an electron sink that stabilizes the charge separated state on the 100-μs time scale. This feature could be exploited in future designs of multiheme cytochromes as components of versatile photosynthetic biohybrid assemblies.
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Clarke, Thomas A., Shirley Fairhurst, David J. Lowe, Nicholas J. Watmough, and Robert R. Eady. "Electron transfer and half-reactivity in nitrogenase." Biochemical Society Transactions 39, no. 1 (January 19, 2011): 201–6. http://dx.doi.org/10.1042/bst0390201.
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Nitrogenase is a globally important enzyme that catalyses the reduction of atmospheric dinitrogen into ammonia and is thus an important part of the nitrogen cycle. The nitrogenase enzyme is composed of a catalytic molybdenum–iron protein (MoFe protein) and a protein containing an [Fe4–S4] cluster (Fe protein) that functions as a dedicated ATP-dependent reductase. The current understanding of electron transfer between these two proteins is based on stopped-flow spectrophotometry, which has allowed the rates of complex formation and electron transfer to be accurately determined. Surprisingly, a total of four Fe protein molecules are required to saturate one MoFe protein molecule, despite there being only two well-characterized Fe-protein-binding sites. This has led to the conclusion that the purified Fe protein is only half-active with respect to electron transfer to the MoFe protein. Studies on the electron transfer between both proteins using rapid-quench EPR confirmed that, during pre-steady-state electron transfer, the Fe protein only becomes half-oxidized. However, stopped-flow spectrophotometry on MoFe protein that had only one active site occupied was saturated by approximately three Fe protein equivalents. These results imply that the Fe protein has a second interaction during the initial stages of mixing that is not involved in electron transfer.
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Kang, S. A., and B. R. Crane. "Effects of interface mutations on association modes and electron-transfer rates between proteins." Proceedings of the National Academy of Sciences 102, no. 43 (October 14, 2005): 15465–70. http://dx.doi.org/10.1073/pnas.0505176102.
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Moser, Christopher C., Christopher C. Page, and P. Leslie Dutton. "Darwin at the molecular scale: selection and variance in electron tunnelling proteins including cytochrome c oxidase." Philosophical Transactions of the Royal Society B: Biological Sciences 361, no. 1472 (July 12, 2006): 1295–305. http://dx.doi.org/10.1098/rstb.2006.1868.
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Biological electron transfer is designed to connect catalytic clusters by chains of redox cofactors. A review of the characterized natural redox proteins with a critical eye for molecular scale measurement of variation and selection related to physiological function shows no statistically significant differences in the protein medium lying between cofactors engaged in physiologically beneficial or detrimental electron transfer. Instead, control of electron tunnelling over long distances relies overwhelmingly on less than 14 Å spacing between the cofactors in a chain. Near catalytic clusters, shorter distances (commonly less than 7 Å) appear to be selected to generate tunnelling frequencies sufficiently high to scale the barriers of multi-electron, bond-forming/-breaking catalysis at physiological rates. We illustrate this behaviour in a tunnelling network analysis of cytochrome c oxidase. In order to surmount the large, thermally activated, adiabatic barriers in the 5–10 kcal mol −1 range expected for H + motion and O 2 reduction at the binuclear centre of oxidase on the 10 3 –10 5 s −1 time-scale of respiration, electron access with a tunnelling frequency of 10 9 or 10 10 s −1 is required. This is provided by selecting closely placed redox centres, such as haem a (6.9 Å) or tyrosine (4.9 Å). A corollary is that more distantly placed redox centres, such as Cu A , cannot rapidly scale the catalytic site barrier, but must send their electrons through more closely placed centres, avoiding direct short circuits that might circumvent proton pumping coupled to haems a to a 3 electron transfer. The selection of distances and energetic barriers directs electron transfer from Cu A to haem a rather than a 3 , without any need for delicate engineering of the protein medium to ‘hard wire’ electron transfer. Indeed, an examination of a large number of oxidoreductases provides no evidence of such naturally selected wiring of electron tunnelling pathways.
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Kuznetsov, Vadim Yu, Emek Blair, Patrick J. Farmer, Thomas L. Poulos, Amanda Pifferitti, and Irina F. Sevrioukova. "The Putidaredoxin Reductase-Putidaredoxin Electron Transfer Complex." Journal of Biological Chemistry 280, no. 16 (February 15, 2005): 16135–42. http://dx.doi.org/10.1074/jbc.m500771200.
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Interaction and electron transfer between putidaredoxin reductase (Pdr) and putidaredoxin (Pdx) fromPseudomonas putidawas studied by molecular modeling, mutagenesis, and stopped flow techniques. Based on the crystal structures of Pdr and Pdx, a complex between the proteins was generated using computer graphics methods. In the model, Pdx is docked above the isoalloxazine ring of FAD of Pdr with the distance between the flavin and [2Fe-2S] of 14.6 Å. This mode of interaction allows Pdx to easily adjust and optimize orientation of its cofactor relative to Pdr. The key residues of Pdx located at the center, Asp38and Trp106, and at the edge of the protein-protein interface, Tyr33and Arg66, were mutated to test the Pdr-Pdx computer model. The Y33F, Y33A, D38N, D38A, R66A, R66E, W106F, W106A, and Δ106 mutations did not affect assembly of the [2Fe-2S] cluster and resulted in a marginal change in the redox potential of Pdx. The electron-accepting ability of Δ106 Pdx was similar to that of the wild-type protein, whereas electron transfer rates from Pdr to other mutants were diminished to various degrees with the smallest and largest effects on the kinetic parameters of the Pdr-to-Pdx electron transfer reaction caused by the Trp106and Tyr33/Arg66substitutions, respectively. Compared with wild-type Pdx, the binding affinity of all studied mutants to Pdr was significantly higher. Experimental results were in agreement with theoretical predictions and suggest that: (i) Pdr-Pdx complex formation is mainly driven by steric complementarity, (ii) bulky side chains of Tyr33, Arg66, and Trp106prevent tight binding of oxidized Pdx and facilitate dissociation of the reduced iron-sulfur protein from Pdr, and (iii) transfer of an electron from FAD to [2Fe-2S] can occur with various orientations between the cofactors through multiple electron transfer pathways that do not involve Trp106but are likely to include Asp38and Cys39.
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Ma, Zhongxin, and Victor L. Davidson. "Ascorbate protects the diheme enzyme, MauG, against self-inflicted oxidative damage by an unusual antioxidant mechanism." Biochemical Journal 474, no. 15 (July 18, 2017): 2563–72. http://dx.doi.org/10.1042/bcj20170349.
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Ascorbate protects MauG from self-inactivation that occurs during the autoreduction of the reactive bis-FeIV state of its diheme cofactor. The mechanism of protection does not involve direct reaction with reactive oxygen species in solution. Instead, it binds to MauG and mitigates oxidative damage that occurs via internal transfer of electrons from amino acid residues within the protein to the high-valent hemes. The presence of ascorbate does not inhibit the natural catalytic reaction of MauG, which catalyzes oxidative post-translational modifications of a substrate protein that binds to the surface of MauG and is oxidized by the high-valent hemes via long-range electron transfer. Ascorbate was also shown to prolong the activity of a P107V MauG variant that is more prone to inactivation. A previously unknown ascorbate peroxidase activity of MauG was characterized with a kcat of 0.24 s−1 and a Km of 2.2 µM for ascorbate. A putative binding site for ascorbate was inferred from inspection of the crystal structure of MauG and comparison with the structure of soybean ascorbate peroxidase with bound ascorbate. The ascorbate bound to MauG was shown to accelerate the rates of both electron transfers to the hemes and proton transfers to hemes which occur during the multistep autoreduction to the diferric state which is accompanied by oxidative damage. A structural basis for these effects is inferred from the putative ascorbate-binding site. This could be a previously unrecognized mechanism by which ascorbate mitigates oxidative damage to heme-dependent enzymes and redox proteins in nature.
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Farooq, Yassar, and Gordon C. K. Roberts. "Kinetics of electron transfer between NADPH-cytochrome P450 reductase and cytochrome P450 3A4." Biochemical Journal 432, no. 3 (November 25, 2010): 485–94. http://dx.doi.org/10.1042/bj20100744.
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We have incorporated CYP3A4 (cytochrome P450 3A4) and CPR (NADPH-cytochrome P450 reductase) into liposomes with a high lipid/protein ratio by an improved method. In the purified proteoliposomes, CYP3A4 binds testosterone with Kd (app)=36±6 μM and Hill coefficient=1.5±0.3, and 75±4% of the CYP3A4 can be reduced by NADPH in the presence of testosterone. Transfer of the first electron from CPR to CYP3A4 was measured by stopped-flow, trapping the reduced CYP3A4 as its Fe(II)–CO complex and measuring the characteristic absorbance change. Rapid electron transfer is observed in the presence of testosterone, with the fast phase, representing 90% of the total absorbance change, having a rate of 14±2 s−1. Measurements of the first electron transfer were performed at various molar ratios of CPR/CYP3A4 in proteoliposomes; the rate was unaffected, consistent with a model in which first electron transfer takes place within a relatively stable CPR–CYP3A4 complex. Steady-state rates of NADPH oxidation and of 6β-hydroxytestosterone formation were also measured as a function of the molar ratio of CPR/CYP3A4 in the proteoliposomes. These rates increased with increasing CPR/CYP3A4 ratio, showing a hyperbolic dependency indicating a Kd (app) of ~0.4 μM. This suggests that the CPR–CYP3A4 complex can dissociate and reform between the first and second electron transfers.
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Qu, Pengxiang, Yuelei Zhao, Rong Wang, Yali Zhang, Lu Li, Jianglin Fan, and Enqi Liu. "Extracellular vesicles derived from donor oviduct fluid improved birth rates after embryo transfer in mice." Reproduction, Fertility and Development 31, no. 2 (2019): 324. http://dx.doi.org/10.1071/rd18203.
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Embryo transfer (ET) is an important procedure for assisted reproduction. However, the relatively lower success rate of ET hampers its application potential. In this study we aimed to elucidate the effects of extracellular vesicles derived from donor oviduct fluid (EDOF) on embryo development after ET. Extracellular vesicles from the oviduct were isolated and purified using ultracentrifugation and identified using transmission electron microscopy, NanoSight, bicinchoninic acid (BCA) protein assay and western blotting. The results revealed that extracellular vesicles were present in donor oviduct fluid in higher concentrations (P<0.05) and contained more proteins (P<0.05) than extracellular vesicles derived from recipient oviduct fluid (EROF). EDOF or EROF were supplemented in an ET medium (ETM) and the results showed that EDOF significantly improved birth rate via resisting apoptosis and promoting differentiation. In conclusion, our study indicated that there are differences in EDOF and EROF and that supplementing EDOF to ETM can improve the efficiency of ET; improved ET efficiency promotes the use of gene editing and benefits assisted reproductive technology and animal welfare.
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Klemme, J. H. "Photoproduction of Hydrogen by Purple Bacteria: A Critical Evaluation of the Rate Limiting Enzymatic Steps." Zeitschrift für Naturforschung C 48, no. 5-6 (June 1, 1993): 482–87. http://dx.doi.org/10.1515/znc-1993-5-613.
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Abstract The enzymatic mechanisms and energetics of nitrogenase-catalyzed photoproduction of hydrogen from organic C-compounds by purple bacteria are discussed in respect to the question of which of the following three enzymes or enzyme systems are rate limiting for H2-production: (a) the nitrogenase-complex; (b) the enzymes and electron transport proteins involved in hydrogen transfer from the organic substrate(s) to nitrogenase; and (c) the system of photosynthetic ATP-regeneration. Calculations of maximum in vivo rates of photosynthetic ATPregeneration (gATP-values derived from growth rates), and of ATP-consumption by nitrogenase- catalyzed H2-production, and comparison of these rates with the specific activities of the enzymes of hydrogen or electron transfer from the C-substrate to the nitrogenase-complex, make it very likely that, in Rhodospirillum rubrum, nitrogenase-catalyzed H2-formation is limited by the availability of ATP and, possibly, of reducing power. In two other nonsulfur purple bacterial species (Rhodobacter capsulatus and Rhodobacter sphaeroides), H2-photoproduction is probably not energy-limited.
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Eiserich, J. P., J. Butler, A. van der Vliet, C. E. Cross, and B. Halliwell. "Nitric oxide rapidly scavenges tyrosine and tryptophan radicals." Biochemical Journal 310, no. 3 (September 15, 1995): 745–49. http://dx.doi.org/10.1042/bj3100745.
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By utilizing a pulse-radiolytic technique, we demonstrate for the first time that the rate constant for the reaction of nitric oxide (.NO) with biologically relevant tyrosine and tryptophan radicals (Tyr. and Trp. respectively) in amino acids, peptides and proteins is of the order of (1-2) x 10(9) M-1.s-1. We also show that .NO effectively interferes with electron-transfer processes between tryptophan and tyrosine residues in proteins subjected to pulse radiolysis. The near diffusion-controlled rates of these reactions, coupled with the increasingly recognized role of protein radicals in enzyme catalysis and oxidative damage, suggest that Tyr. and Trp. are likely and important targets for .NO generated in vivo.
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Onuchic, José N., Chigusa Kobayashi, Osamu Miyashita, Patricia Jennings, and Kim K. Baldridge. "Exploring biomolecular machines: energy landscape control of biological reactions." Philosophical Transactions of the Royal Society B: Biological Sciences 361, no. 1472 (July 14, 2006): 1439–43. http://dx.doi.org/10.1098/rstb.2006.1876.
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For almost 15 years, our Pathway model has been the most powerful model in terms of predicting the tunnelling mechanism for electron transfer (ET) in biological systems, particularly proteins. Going beyond the conventional Pathway models, we have generalized our method to understand how protein dynamics modulate not only the Franck–Condon factor, but also the tunnelling matrix element. We have demonstrated that when interference among pathways modulates the electron tunnelling interactions in proteins (particularly destructive interference), dynamical effects are of critical importance. Tunnelling can be controlled by protein conformations that lie far from equilibrium—those that minimize the effect of destructive interference during tunnelling, for example. In the opposite regime, electron tunnelling is mediated by one (or a few) constructively interfering pathway tubes and dynamical effects are modest. This new mechanism for dynamical modulation of the ET rate has been able to explain and/or predict several rates that were later confirmed by experiment. However, thermal fluctuations can also affect these molecular machines in many other ways. For example, we show how global transformations, which control protein functions such as allostery, may involve large-scale motion and possibly partial unfolding during the reaction event.
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Blank, Carrine E. "Low Rates of Lateral Gene Transfer among Metabolic Genes Define the Evolving Biogeochemical Niches of Archaea through Deep Time." Archaea 2012 (2012): 1–23. http://dx.doi.org/10.1155/2012/843539.
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Phylogenomic analyses of archaeal genome sequences are providing windows into the group’s evolutionary past, even though most archaeal taxa lack a conventional fossil record. Here, phylogenetic analyses were performed using key metabolic genes that define the metabolic niche of microorganisms. Such genes are generally considered to have undergone high rates of lateral gene transfer. Many gene sequences formed clades that were identical, or similar, to the tree constructed using large numbers of genes from the stable core of the genome. Surprisingly, such lateral transfer events were readily identified and quantifiable, occurring only a relatively small number of times in the archaeal domain of life. By placing gene acquisition events into a temporal framework, the rates by which new metabolic genes were acquired can be quantified. The highest lateral transfer rates were among cytochrome oxidase genes that use oxygen as a terminal electron acceptor (with a total of 12–14 lateral transfer events, or 3.4–4.0 events per billion years, across the entire archaeal domain). Genes involved in sulfur or nitrogen metabolism had much lower rates, on the order of one lateral transfer event per billion years. This suggests that lateral transfer rates of key metabolic proteins are rare and not rampant.
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LLOYD, Jon R., Ching LEANG, Allison L. HODGES MYERSON, Maddalena V. COPPI, Stacey CUIFO, Barb METHE, Steven J. SANDLER, and Derek R. LOVLEY. "Biochemical and genetic characterization of PpcA, a periplasmic c-type cytochrome in Geobacter sulfurreducens." Biochemical Journal 369, no. 1 (January 1, 2003): 153–61. http://dx.doi.org/10.1042/bj20020597.
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A 9.6kDa periplasmic c-type cytochrome, designated PpcA, was purified from the Fe(III)-reducing bacterium Geobacter sulfurreducens and characterized. The purified protein is basic (pI 9.5), contains three haems and has an N-terminal amino acid sequence closely related to those of the previously described trihaem c7 cytochromes of Geobacter metallireducens and Desulfuromonas acetoxidans. The gene encoding PpcA was identified from the G. sulfurreducens genome using the N-terminal sequence, and encodes a protein of 71 amino acids (molecular mass 9.58kDa) with 49% identity to the c7 cytochrome of D. acetoxidans. In order to determine the physiological role of PpcA, a knockout mutant was prepared with a single-step recombination method. Acetate-dependent Fe(III) reduction was significantly inhibited in both growing cultures and cell suspensions of the mutant. When ppcA was expressed in trans, the full capacity for Fe(III) reduction with acetate was restored. The transfer of electrons from acetate to anthraquinone 2,6-disulphonate (AQDS; a humic acid analogue) and to U(VI) was also compromised in the mutant, but acetate-dependent reduction of fumarate was not altered. The rates of reduction of Fe(III), AQDS, U(VI) and fumarate were also the same in the wild type and ppcA mutant when hydrogen was supplied as the electron donor. When taken together with previous studies on other electron transport proteins in G. sulfurreducens, these results suggest that PpcA serves as an intermediary electron carrier from acetate to terminal Fe(III) reductases in the outer membrane, and is also involved in the transfer of electrons from acetate to U(VI) and humics.
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Orłowski, Rafał, John A. Clark, James B. Derr, Eli M. Espinoza, Maximilian F. Mayther, Olga Staszewska-Krajewska, Jay R. Winkler, et al. "Role of intramolecular hydrogen bonds in promoting electron flow through amino acid and oligopeptide conjugates." Proceedings of the National Academy of Sciences 118, no. 11 (March 11, 2021): e2026462118. http://dx.doi.org/10.1073/pnas.2026462118.
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Elucidating the factors that control charge transfer rates in relatively flexible conjugates is of importance for understanding energy flows in biology as well as assisting the design and construction of electronic devices. Here, we report ultrafast electron transfer (ET) and hole transfer (HT) between a corrole (Cor) donor linked to a perylene-diimide (PDI) acceptor by a tetrameric alanine (Ala)4. Selective photoexcitation of the donor and acceptor triggers subpicosecond and picosecond ET and HT. Replacement of the (Ala)4 linker with either a single alanine or phenylalanine does not substantially affect the ET and HT kinetics. We infer that electronic coupling in these reactions is not mediated by tetrapeptide backbone nor by direct donor–acceptor interactions. Employing a combination of NMR, circular dichroism, and computational studies, we show that intramolecular hydrogen bonding brings the donor and the acceptor into proximity in a “scorpion-shaped” molecular architecture, thereby accounting for the unusually high ET and HT rates. Photoinduced charge transfer relies on a (Cor)NH…O=C–NH…O=C(PDI) electronic-coupling pathway involving two pivotal hydrogen bonds and a central amide group as a mediator. Our work provides guidelines for construction of effective donor–acceptor assemblies linked by long flexible bridges as well as insights into structural motifs for mediating ET and HT in proteins.
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Fernandes, Tomás M., Leonor Morgado, David L. Turner, and Carlos A. Salgueiro. "Protein Engineering of Electron Transfer Components from Electroactive Geobacter Bacteria." Antioxidants 10, no. 6 (May 25, 2021): 844. http://dx.doi.org/10.3390/antiox10060844.
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Electrogenic microorganisms possess unique redox biological features, being capable of transferring electrons to the cell exterior and converting highly toxic compounds into nonhazardous forms. These microorganisms have led to the development of Microbial Electrochemical Technologies (METs), which include applications in the fields of bioremediation and bioenergy production. The optimization of these technologies involves efforts from several different disciplines, ranging from microbiology to materials science. Geobacter bacteria have served as a model for understanding the mechanisms underlying the phenomenon of extracellular electron transfer, which is highly dependent on a multitude of multiheme cytochromes (MCs). MCs are, therefore, logical targets for rational protein engineering to improve the extracellular electron transfer rates of these bacteria. However, the presence of several heme groups complicates the detailed redox characterization of MCs. In this Review, the main characteristics of electroactive Geobacter bacteria, their potential to develop microbial electrochemical technologies and the main features of MCs are initially highlighted. This is followed by a detailed description of the current methodologies that assist the characterization of the functional redox networks in MCs. Finally, it is discussed how this information can be explored to design optimal Geobacter-mutated strains with improved capabilities in METs.
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Thorneley, R. N. F., and G. A. Ashby. "Oxidation of nitrogenase iron protein by dioxygen without inactivation could contribute to high respiration rates of Azotobacter species and facilitate nitrogen fixation in other aerobic environments." Biochemical Journal 261, no. 1 (July 1, 1989): 181–87. http://dx.doi.org/10.1042/bj2610181.
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The kinetics of oxidation of the Fe proteins of nitrogenases from Klebsiella pneumoniae (Kp2) and Azotobacter chroococcum (Ac2) by O2 and H2O2 have been studied by stopped-flow spectrophotometry at 23 degrees C, pH 7.4. With excess O2, one-electron oxidation of Kp2 and Ac2 and their 2 MgATP or 2 MgADP bound forms occurs with rate constants (k) in the range 5.3 x 10(3) M-1.S-1 to 1.6 x 10(5) M-1.S-1. A linear correlation between log k and the mid-point potentials (Em) of these protein species indicates that the higher rates of electron transfer from the Ac2 species are due to the differences in Em of the 4Fe-4S cluster. The reaction of Ac2(MgADP)2 with O2 is sufficiently rapid for it to contribute significantly to the high respiration rate of Azotobacter under N2-fixing conditions and may represent a new respiratory pathway. Excess O2 rapidly inactivates Ac2(MgADP)2 and Kp2(MgADP)2; however, when these protein species are in greater than 4-fold molar excess over the concentration of O2, 4 equivalents of protein are oxidized with no loss of activity. The kinetics of this reaction suggest that H2O2 is an intermediate in the reduction of O2 to 2 H2O by nitrogenase Fe proteins and imply a role for catalase or peroxidase in the mechanism of protection of nitrogenase from O2-induced inactivation.
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Marzolf, Daniel R., Aidan M. McKenzie, Matthew C. O’Malley, Nina S. Ponomarenko, Coleman M. Swaim, Tyler J. Brittain, Natalie L. Simmons, et al. "Mimicking Natural Photosynthesis: Designing Ultrafast Photosensitized Electron Transfer into Multiheme Cytochrome Protein Nanowires." Nanomaterials 10, no. 11 (October 28, 2020): 2143. http://dx.doi.org/10.3390/nano10112143.
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Efficient nanomaterials for artificial photosynthesis require fast and robust unidirectional electron transfer (ET) from photosensitizers through charge-separation and accumulation units to redox-active catalytic sites. We explored the ultrafast time-scale limits of photo-induced charge transfer between a Ru(II)tris(bipyridine) derivative photosensitizer and PpcA, a 3-heme c-type cytochrome serving as a nanoscale biological wire. Four covalent attachment sites (K28C, K29C, K52C, and G53C) were engineered in PpcA enabling site-specific covalent labeling with expected donor-acceptor (DA) distances of 4–8 Å. X-ray scattering results demonstrated that mutations and chemical labeling did not disrupt the structure of the proteins. Time-resolved spectroscopy revealed three orders of magnitude difference in charge transfer rates for the systems with otherwise similar DA distances and the same number of covalent bonds separating donors and acceptors. All-atom molecular dynamics simulations provided additional insight into the structure-function requirements for ultrafast charge transfer and the requirement of van der Waals contact between aromatic atoms of photosensitizers and hemes in order to observe sub-nanosecond ET. This work demonstrates opportunities to utilize multi-heme c-cytochromes as frameworks for designing ultrafast light-driven ET into charge-accumulating biohybrid model systems, and ultimately for mimicking the photosynthetic paradigm of efficiently coupling ultrafast, light-driven electron transfer chemistry to multi-step catalysis within small, experimentally versatile photosynthetic biohybrid assemblies.
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Allen, John F., and Thomas Pfannschmidt. "Balancing the two photosystems: photosynthetic electron transfer governs transcription of reaction centre genes in chloroplasts." Philosophical Transactions of the Royal Society of London. Series B: Biological Sciences 355, no. 1402 (October 29, 2000): 1351–59. http://dx.doi.org/10.1098/rstb.2000.0697.
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Chloroplasts are cytoplasmic organelles whose primary function is photosynthesis, but which also contain small, specialized and quasi–autonomous genetic systems. In photosynthesis, two energy converting photosystems are connected, electrochemically, in series. The connecting electron carriers are oxidized by photosystem I (PS I) and reduced by photosystem II (PS II). It has recently been shown that the oxidation–reduction state of one connecting electron carrier, plastoquinone, controls transcription of chloroplast genes for reaction centre proteins of the two photosystems. The control counteracts the imbalance in electron transport that causes it: oxidized plastoquinone induces PS II and represses PS I; reduced plastoquinone induces PS I and represses PS II. This complementarity is observed both in vivo , using light favouring one or other photosystem, and in vitro , when site–specific electron transport inhibitors are added to transcriptionally and photosynthetically active chloroplasts. There is thus a transcriptional level of control that has a regulatory function similar to that of purely post–translational ‘state transitions’ in which the redistribution of absorbed excitation energy between photosystems is mediated by thylakoid membrane protein phosphorylation. The changes in rates of transcription that are induced by spectral changes in vivo can be detected even before the corresponding state transitions are complete, suggesting the operation of a branched pathway of redox signal transduction. These findings suggest a mechanism for adjustment of photosystem stoichiometry in which initial events involve a sensor of the redox state of plastoquinone, and may thus be the same as the initial events of state transitions. Redox control of chloroplast transcription is also consistent with the proposal that a direct regulatory coupling between electron transport and gene expression determines the function and composition of the chloroplast's extra–nuclear genetic system.
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Arnesano, Fabio, Lucia Banci, and Mario Piccioli. "NMR structures of paramagnetic metalloproteins." Quarterly Reviews of Biophysics 38, no. 2 (May 2005): 167–219. http://dx.doi.org/10.1017/s0033583506004161.
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1. Introduction 1681.1 Genomic annotation of metalloproteins 1681.2 Why NMR structures? 1681.3 Why paramagnetic metalloproteins? 1692. General theory 1702.1 Nuclear and electron spins 1702.2 Hyperfine coupling 1712.3 The effect of the hyperfine coupling on the NMR shift: the hyperfine shift 1732.4 The effect of the hyperfine coupling on nuclear relaxation 1742.5 Interplay between electron spin properties and features of the NMR spectra 1783. Paramagnetism-based structural restraints 1803.1 Contact shifts and relaxation rates as restraints 1813.2 Locating the metal ion within the protein frame: pseudocontact shifts 1843.3 Cross-correlation rates 1863.4 Residual dipolar couplings 1883.5 Interplay between different restraints 1904. NMR without1H detection 1914.1 The protocol for 13C-detected protonless assignment of backbone and side-chains 1944.2 Protonless heteronuclear NMR experiments tailored to paramagnetic systems 1965. The use of lanthanides as paramagnetic probes 1986. The case of Cu(II) proteins 2027. Perspectives 2088. Acknowledgments 2099. References 209Metalloproteins represent a large share of the proteome and many of them contain paramagnetic metal ions. The knowledge, at atomic resolution, of their structure in solution is important to understand processes in which they are involved, such as electron transfer mechanisms, enzymatic reactions, metal homeostasis and metal trafficking, as well as interactions with their partners. Formerly considered as unfeasible, the first structure in solution by nuclear magnetic resonance (NMR) of a paramagnetic protein was obtained in 1994. Methodological and instrumental advancements pursued over the last decade are such that NMR structure of paramagnetic proteins may be now routinely obtained. We focus here on approaches and problems related to the structure determination of paramagnetic proteins in solution through NMR spectroscopy. After a survey of the background theory, we show how the effects produced by the presence of a paramagnetic metal ion on the NMR parameters, which are in many cases deleterious for the detection of NMR spectra, can be overcome and turned into an additional source of structural restraints. We also briefly address features and perspectives given by the use of 13C-detected protonless NMR spectroscopy for proteins in solution. The structural information obtained through the exploitation of a paramagnetic center are discussed for some Cu2+-binding proteins and for Ca2+-binding proteins, where the replacement of a diamagnetic metal ion with suitable paramagnetic metal ions suggests novel approaches to the structural characterization of proteins containing diamagnetic and NMR-silent metal ions.
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Bergström, J., R. R. Eady, and R. N. F. Thorneley. "The vanadium- and molybdenum-containing nitrogenases of Azotobacter chroococcum. Comparison of mid-point potentials and kinetics of reduction by sodium dithionite of the iron proteins with bound magnesium adenosine 5′-diphosphate." Biochemical Journal 251, no. 1 (April 1, 1988): 165–69. http://dx.doi.org/10.1042/bj2510165.
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The mid-point potentials of the Fe protein components (Ac2 and Ac2* respectively) of the Mo nitrogenase and V nitrogenase from Azotobacter chroococcum were determined in the presence of MgADP to be −450 mV (NHE) [Ac2(MgADP)2-Ac2*ox.(MgADP)2 couple] and −463 mV (NHE) [Ac2* (MgADP)2-Ac2*ox.(ADP)2 couple] at 23 degrees C at pH 7.2. These values are consistent with a flavodoxin characterized by Deistung & Thorneley [(1986) Biochem. J. 239, 69-75] with Em = −522 mV (NHE) being an effective electron donor to both the Mo nitrogenase and the V nitrogenase in vivo. Ac2*ox.(MgADP)2 and Ac2*ox.(MgADP)2 were reduced by SO2.- (formed by the predissociation of dithionite ion, S2O4(2-)) at similar rates, k = 4.7 × 10(6) +/- 0.5 × 10(6) M-1.s-1 and 3.2 × 10(6) +/- 0.2 × 10(6) M-1.s-1 respectively, indicating structural homology at the electron-transfer site associated with the [4Fe-4S] centre in these proteins.
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Ishii, Shun'ichi, Kazuya Watanabe, Soichi Yabuki, Bruce E. Logan, and Yuji Sekiguchi. "Comparison of Electrode Reduction Activities of Geobacter sulfurreducens and an Enriched Consortium in an Air-Cathode Microbial Fuel Cell." Applied and Environmental Microbiology 74, no. 23 (October 3, 2008): 7348–55. http://dx.doi.org/10.1128/aem.01639-08.
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ABSTRACT An electricity-generating bacterium, Geobacter sulfurreducens PCA, was inoculated into a single-chamber, air-cathode microbial fuel cell (MFC) in order to determine the maximum electron transfer rate from bacteria to the anode. To create anodic reaction-limiting conditions, where electron transfer from bacteria to the anode is the rate-limiting step, anodes with electrogenic biofilms were reduced in size and tests were conducted using anodes of six different sizes. The smallest anode (7 cm2, or 1.5 times larger than the cathode) achieved an anodic reaction-limiting condition as a result of a limited mass of bacteria on the electrode. Under these conditions, the limiting current density reached a maximum of 1,530 mA/m2, and power density reached a maximum of 461 mW/m2. Per-biomass efficiency of the electron transfer rate was constant at 32 fmol cell−1 day−1 (178 μmol g of protein−1 min−1), a rate comparable to that with solid iron as the electron acceptor but lower than rates achieved with fumarate or soluble iron. In comparison, an enriched electricity-generating consortium reached 374 μmol g of protein−1 min−1 under the same conditions, suggesting that the consortium had a much greater capacity for electrode reduction. These results demonstrate that per-biomass electrode reduction rates (calculated by current density and biomass density on the anode) can be used to help make better comparisons of electrogenic activity in MFCs.
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Margulis, Ketty, Hagit Zer, Hagar Lis, Hanan Schoffman, Omer Murik, Ginga Shimakawa, Anja Krieger-Liszkay, and Nir Keren. "Over Expression of the Cyanobacterial Pgr5-Homologue Leads to Pseudoreversion in a Gene Coding for a Putative Esterase in Synechocystis 6803." Life 10, no. 9 (September 3, 2020): 174. http://dx.doi.org/10.3390/life10090174.
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Pgr5 proteins play a major direct role in cyclic electron flow paths in plants and eukaryotic phytoplankton. The genomes of many cyanobacterial species code for Pgr5-like proteins but their function is still uncertain. Here, we present evidence that supports a link between the Synechocystis sp. PCC6803 Pgr5-like protein and the regulation of intracellular redox balance. The knockout strain, pgr5KO, did not display substantial phenotypic response under our experimental conditions, confirming results obtained in earlier studies. However, the overexpression strain, pgr5OE, accumulated 2.5-fold more chlorophyll than the wild type and displayed increased content of photosystems matching the chlorophyll increase. As a result, electron transfer rates through the photosynthetic apparatus of pgr5OE increased, as did the amount of energy stored as glycogen. While, under photoautotrophic conditions, this metabolic difference had only minor effects, under mixotrophic conditions, pgr5OE cultures collapsed. Interestingly, this specific phenotype of pgr5OE mutants displayed a tendency for reverting, and cultures which previously collapsed in the presence of glucose were now able to survive. DNA sequencing of a pgr5OE strain revealed a second site suppression mutation in slr1916, a putative esterase associated with redox regulation. The phenotype of the slr1916 knockout is very similar to that of the strain reported here and to that of the pmgA regulator knockout. These data demonstrate that, in Synechocystis 6803, there is strong selection against overexpression of the Pgr5-like protein. The pseudoreversion event in a gene involved in redox regulation suggests a connection of the Pgr5-like protein to this network.
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Bennett, James P., and Isaac G. Onyango. "Energy, Entropy and Quantum Tunneling of Protons and Electrons in Brain Mitochondria: Relation to Mitochondrial Impairment in Aging-Related Human Brain Diseases and Therapeutic Measures." Biomedicines 9, no. 2 (February 22, 2021): 225. http://dx.doi.org/10.3390/biomedicines9020225.
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Adult human brains consume a disproportionate amount of energy substrates (2–3% of body weight; 20–25% of total glucose and oxygen). Adenosine triphosphate (ATP) is a universal energy currency in brains and is produced by oxidative phosphorylation (OXPHOS) using ATP synthase, a nano-rotor powered by the proton gradient generated from proton-coupled electron transfer (PCET) in the multi-complex electron transport chain (ETC). ETC catalysis rates are reduced in brains from humans with neurodegenerative diseases (NDDs). Declines of ETC function in NDDs may result from combinations of nitrative stress (NS)–oxidative stress (OS) damage; mitochondrial and/or nuclear genomic mutations of ETC/OXPHOS genes; epigenetic modifications of ETC/OXPHOS genes; or defects in importation or assembly of ETC/OXPHOS proteins or complexes, respectively; or alterations in mitochondrial dynamics (fusion, fission, mitophagy). Substantial free energy is gained by direct O2-mediated oxidation of NADH. Traditional ETC mechanisms require separation between O2 and electrons flowing from NADH/FADH2 through the ETC. Quantum tunneling of electrons and much larger protons may facilitate this separation. Neuronal death may be viewed as a local increase in entropy requiring constant energy input to avoid. The ATP requirement of the brain may partially be used for avoidance of local entropy increase. Mitochondrial therapeutics seeks to correct deficiencies in ETC and OXPHOS.
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Bond, Daniel R., and Derek R. Lovley. "Electricity Production by Geobacter sulfurreducens Attached to Electrodes." Applied and Environmental Microbiology 69, no. 3 (March 2003): 1548–55. http://dx.doi.org/10.1128/aem.69.3.1548-1555.2003.
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ABSTRACT Previous studies have suggested that members of the Geobacteraceae can use electrodes as electron acceptors for anaerobic respiration. In order to better understand this electron transfer process for energy production, Geobacter sulfurreducens was inoculated into chambers in which a graphite electrode served as the sole electron acceptor and acetate or hydrogen was the electron donor. The electron-accepting electrodes were maintained at oxidizing potentials by connecting them to similar electrodes in oxygenated medium (fuel cells) or to potentiostats that poised electrodes at +0.2 V versus an Ag/AgCl reference electrode (poised potential). When a small inoculum of G. sulfurreducens was introduced into electrode-containing chambers, electrical current production was dependent upon oxidation of acetate to carbon dioxide and increased exponentially, indicating for the first time that electrode reduction supported the growth of this organism. When the medium was replaced with an anaerobic buffer lacking nutrients required for growth, acetate-dependent electrical current production was unaffected and cells attached to these electrodes continued to generate electrical current for weeks. This represents the first report of microbial electricity production solely by cells attached to an electrode. Electrode-attached cells completely oxidized acetate to levels below detection (<10 μM), and hydrogen was metabolized to a threshold of 3 Pa. The rates of electron transfer to electrodes (0.21 to 1.2 μmol of electrons/mg of protein/min) were similar to those observed for respiration with Fe(III) citrate as the electron acceptor (Eo′ =+0.37 V). The production of current in microbial fuel cell (65 mA/m2 of electrode surface) or poised-potential (163 to 1,143 mA/m2) mode was greater than what has been reported for other microbial systems, even those that employed higher cell densities and electron-shuttling compounds. Since acetate was completely oxidized, the efficiency of conversion of organic electron donor to electricity was significantly higher than in previously described microbial fuel cells. These results suggest that the effectiveness of microbial fuel cells can be increased with organisms such as G. sulfurreducens that can attach to electrodes and remain viable for long periods of time while completely oxidizing organic substrates with quantitative transfer of electrons to an electrode.
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Jansen, Marcel A. K., and Klaus Pfister. "Conserved Kinetics at the Reducing Side of Reaction-Center II in Photosynthetic Organisms; Changed Kinetics in Triazine-Resistant Weeds." Zeitschrift für Naturforschung C 45, no. 5 (May 1, 1990): 441–45. http://dx.doi.org/10.1515/znc-1990-0522.
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The decay of chlorophyll variable fluorescence after a “single turnover” flash is generally assumed to represent the reoxidation of the reduced quinone Qa. We have observed that the kinetics of this decay are very similar in a wide variety of species. Comparing 28 different species, we found an average half decay time of 314 ± 46 μsec. No systematic correlations were found between the decay rate and biochemical or physiological specializations such as C2, C4 or CAM. This indicates that structural as well as functional factors controlling photosystem II electron transfer between Qa and Qb are highly conserved. Apparently, the freedom for natural structural variations in this region is very limited. Triazine resistant plants, characterized by an altered amino acid sequence of the D1 protein, have clearly decreased rates of Qa/Qb electron transfer. We found an average half decay time of 946 ± 100 (isec (5 species). However, this three-fold decrease is much less than previously reported. Therefore, if alterations of photosystem II electron transfer efficiency contributes to an often reported reduction of “ecological fitness”, this contribution is smaller than was hitherto assumed.
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Sarti, P., G. Antonini, F. Malatesta, and M. Brunori. "Respiratory control in cytochrome oxidase vesicles is correlated with the rate of internal electron transfer." Biochemical Journal 284, no. 1 (May 15, 1992): 123–27. http://dx.doi.org/10.1042/bj2840123.
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Cytochrome c oxidase, after reconstitution into phospholipid vesicles, displays respiratory control. This appears as an inhibition of substrate oxidation (cytochrome c) or reduction (O2) rates which, in the first few turnovers, can be largely removed upon addition of valinomycin, a specific K+ carrier. We report experiments designed to measure directly the internal electron transfer leading to the reduction of cytochrome a3/CuB, in the presence and the absence of a membrane potential. The results suggest that, after the complete oxidation and partial re-reduction of the protein, electron transfer to the binuclear site is valinomycin-sensitive, i.e. is inhibited by the membrane potential. The first-order rate constants calculated in the absence and presence of valinomycin were 0.5-0.6 and 5-6 s-1 respectively. Kinetic analysis of the reduction process is consistent with the conclusion that the membrane potential is below the critical threshold until the first electron is transferred to the cytochrome a3/CuB site. Furthermore, the respiratory control ratio obtained from the dependence of the internal electron transfer rate constant on valinomycin is always higher (by factor of 2) than that measured under turnover conditions either polarographically or spectrophotometrically. Two possible interpretations of this discrepancy are discussed.
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Brooks, H. B., and V. L. Davidson. "A method for extracting rate constants from initial rates of stopped-flow kinetic data: application to a physiological electron-transfer reaction." Biochemical Journal 294, no. 1 (August 15, 1993): 211–13. http://dx.doi.org/10.1042/bj2940211.
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The most commonly used methods for analysis of stopped-flow kinetic data require performing a series of measurements in which one reactant is varied at concentrations significantly greater than the concentration of the other reactant. For enzyme-catalysed reactions this may not be possible, because the dissociation constants for the enzyme-substrate complex are often of the same order of magnitude as the high concentrations of enzyme that must frequently be used in stopped-flow studies. An alternative method of data analysis is presented which allows the determination of microscopic rate constants from initial rates of stopped-flow kinetic data in which substrate is varied in a range of concentrations approximately the same as the enzyme. This method also provides a simple and accurate method for determining k4, the rate of the reverse reaction. This method has been used to describe a physiological electron transfer reaction between a quinoprotein, methylamine dehydrogenase, and a copper protein, amicyanin. At 20 degrees C, the rate of the electron-transfer reaction from methylamine dehydrogenase to amicyanin was 24 s-1, and the dissociation constant for complex-formation was 1.9 microM.
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Navarro, Ana, and Alberto Boveris. "The mitochondrial energy transduction system and the aging process." American Journal of Physiology-Cell Physiology 292, no. 2 (February 2007): C670—C686. http://dx.doi.org/10.1152/ajpcell.00213.2006.
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Aged mammalian tissues show a decreased capacity to produce ATP by oxidative phosphorylation due to dysfunctional mitochondria. The mitochondrial content of rat brain and liver is not reduced in aging and the impairment of mitochondrial function is due to decreased rates of electron transfer by the selectively diminished activities of complexes I and IV. Inner membrane H+ impermeability and F1-ATP synthase activity are only slightly affected by aging. Dysfunctional mitochondria in aged rodents are characterized, besides decreased electron transfer and O2 uptake, by an increased content of oxidation products of phospholipids, proteins and DNA, a decreased membrane potential, and increased size and fragility. Free radical-mediated oxidations are determining factors of mitochondrial dysfunction and turnover, cell apoptosis, tissue function, and lifespan. Inner membrane enzyme activities, such as those of complexes I and IV and mitochondrial nitric oxide synthase, decrease upon aging and afford aging markers. The activities of these three enzymes in mice brain are linearly correlated with neurological performance, as determined by the tightrope and the T-maze tests. The same enzymatic activities correlated positively with mice survival and negatively with the mitochondrial content of lipid and protein oxidation products. Conditions that increase survival, as vitamin E dietary supplementation, caloric restriction, high spontaneous neurological activity, and moderate physical exercise, ameliorate mitochondrial dysfunction in aged brain and liver. The pleiotropic signaling of mitochondrial H2O2 and nitric oxide diffusion to the cytosol seems modified in aged animals and to contribute to the decreased mitochondrial biogenesis in old animals.
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Smith, Jessica A., Pier-Luc Tremblay, Pravin Malla Shrestha, Oona L. Snoeyenbos-West, Ashley E. Franks, Kelly P. Nevin, and Derek R. Lovley. "Going Wireless: Fe(III) Oxide Reduction without Pili by Geobacter sulfurreducens Strain JS-1." Applied and Environmental Microbiology 80, no. 14 (May 9, 2014): 4331–40. http://dx.doi.org/10.1128/aem.01122-14.
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ABSTRACTPrevious studies have suggested that the conductive pili ofGeobacter sulfurreducensare essential for extracellular electron transfer to Fe(III) oxides and for optimal long-range electron transport through current-producing biofilms. The KN400 strain ofG. sulfurreducensreduces poorly crystalline Fe(III) oxide more rapidly than the more extensively studied DL-1 strain. Deletion of the gene encoding PilA, the structural pilin protein, in strain KN400 inhibited Fe(III) oxide reduction. However, low rates of Fe(III) reduction were detected after extended incubation (>30 days) in the presence of Fe(III) oxide. After seven consecutive transfers, the PilA-deficient strain adapted to reduce Fe(III) oxide as fast as the wild type. Microarray, whole-genome resequencing, proteomic, and gene deletion studies indicated that this adaptation was associated with the production of larger amounts of thec-type cytochrome PgcA, which was released into the culture medium. It is proposed that the extracellular cytochrome acts as an electron shuttle, promoting electron transfer from the outer cell surface to Fe(III) oxides. The adapted PilA-deficient strain competed well with the wild-type strain when both were grown together on Fe(III) oxide. However, when 50% of the culture medium was replaced with fresh medium every 3 days, the wild-type strain outcompeted the adapted strain. A possible explanation for this is that the necessity to produce additional PgcA, to replace the PgcA being continually removed, put the adapted strain at a competitive disadvantage, similar to the apparent selection against electron shuttle-producing Fe(III) reducers in many anaerobic soils and sediments. Despite increased extracellular cytochrome production, the adapted PilA-deficient strain produced low levels of current, consistent with the concept that long-range electron transport throughG. sulfurreducensbiofilms is more effective via pili.
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Subramanian, Poorna, Sahand Pirbadian, Mohamed Y. El-Naggar, and Grant J. Jensen. "Ultrastructure of Shewanella oneidensis MR-1 nanowires revealed by electron cryotomography." Proceedings of the National Academy of Sciences 115, no. 14 (March 19, 2018): E3246—E3255. http://dx.doi.org/10.1073/pnas.1718810115.
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Bacterial nanowires have garnered recent interest as a proposed extracellular electron transfer (EET) pathway that links the bacterial electron transport chain to solid-phase electron acceptors away from the cell. Recent studies showed that Shewanella oneidensis MR-1 produces outer membrane (OM) and periplasmic extensions that contain EET components and hinted at their possible role as bacterial nanowires. However, their fine structure and distribution of cytochrome electron carriers under native conditions remained unclear, making it difficult to evaluate the potential electron transport (ET) mechanism along OM extensions. Here, we report high-resolution images of S. oneidensis OM extensions, using electron cryotomography (ECT). We developed a robust method for fluorescence light microscopy imaging of OM extension growth on electron microscopy grids and used correlative light and electron microscopy to identify and image the same structures by ECT. Our results reveal that S. oneidensis OM extensions are dynamic chains of interconnected outer membrane vesicles (OMVs) with variable dimensions, curvature, and extent of tubulation. Junction densities that potentially stabilize OMV chains are seen between neighboring vesicles in cryotomograms. By comparing wild type and a cytochrome gene deletion mutant, our ECT results provide the likely positions and packing of periplasmic and outer membrane proteins consistent with cytochromes. Based on the observed cytochrome packing density, we propose a plausible ET path along the OM extensions involving a combination of direct hopping and cytochrome diffusion. A mean-field calculation, informed by the observed ECT cytochrome density, supports this proposal by revealing ET rates on par with a fully packed cytochrome network.
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Best, Stephen, M. Tauhid Islam, Christopher Chantler, and Jay Bourke. "Redox modulation of Low-Volume (100 µL) Solutions for XAFS measurements." Acta Crystallographica Section A Foundations and Advances 70, a1 (August 5, 2014): C1523. http://dx.doi.org/10.1107/s2053273314084769.
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A key to the understanding of transition metal catalysis is a detailed knowledge of the changes in coordination environment that accompany a change in redox state. The capacity of a given metal complex to support the high rates of electron transfer needed for effective catalysis is strongly dependent on the magnitude of structural reorganization coupled to the redox step. The ability of the ligand to control the dynamics of electron transfer is beautifully illustrated by copper redox proteins such as plastocyanin.[1] The polypeptide-imposed constraints on the environment at the coordination site of the metal minimize the structural change attendant on interconversion between the CuI and CuII redox states of the metal, facilitating fast electron transfer. Further, unravelling the molecular details of enzyme catalysis often hinges on knowledge of the structural changes attendant on oxidation or reduction. XAFS can provide the key structural information for reactive or unstable redox states of biological and abiological molecules. Our research has mostly centred on the use of a combination of spectroscopic and computational techniques to reveal the chemistry associated with dihydrogen activation in diiron compounds related to [FeFe]-hydrogenases [2], where a combination of XAFS, IR spectroscopy and theory can provide reliable structural information. Sampling of such species can provide a comparable challenge to spectral analysis – an issue made more difficult in cases where the quantity of sample is limited. We have developed low-volume electrosynthesis cells suitable for the study of electrogenerated species where the total volume of solution required for XAS data collection is of order 100 µL [3]. The design and operation of cells designed to allow freeze quenching and tow-temperature spectral collection or RT on-line measurement will be described.
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MOORE, Geoffrey R., Mark C. COX, David CROWE, Michael J. OSBORNE, Federico I. ROSELL, Jordi BUJONS, Paul D. BARKER, Marcia R. MAUK, and A. Grant MAUK. "Nε,Nε-Dimethyl-lysine cytochrome c as an NMR probe for lysine involvement in protein–protein complex formation." Biochemical Journal 332, no. 2 (June 1, 1998): 439–49. http://dx.doi.org/10.1042/bj3320439.
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The reductively dimethylated derivatives of horse and yeast iso-1-ferricytochromes c have been prepared and characterized for use as NMR probes of the complexes formed by cytochrome c with bovine liver cytochrome b5 and yeast cytochrome c peroxidase. The electrostatic properties and structures of the derivatized cytochromes are not significantly perturbed by the modifications; neither are the electrostatics of protein–protein complex formation or rates of interprotein electron transfer. Two-dimensional 1H–13C NMR spectroscopy of the complexes formed by the derivatized cytochromes with cytochrome b5 and cytochrome c peroxidase has been used to investigate the number and identity of lysine residues of cytochrome c that are involved in interprotein interactions of cytochrome c. The NMR data are incompatible with simple static models proposed previously for the complexes formed by these proteins, but are consistent with the presence of multiple, interconverting complexes of comparable stability, consistent with studies employing Brownian dynamics to model the complexes. The NMR characteristics of the Nε,Nε-dimethyl-lysine groups, their chemical shift dispersion, oxidation state and temperature dependences and the possibility of chemical exchange phenomena are discussed with relevance to the utility of Nε,Nε-dimethyl-lysine's being a generally useful derivative for characterizing protein–protein complexes.
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Osorio, Héctor, Stefanie Mangold, Yann Denis, Ivan Ñancucheo, Mario Esparza, D. Barrie Johnson, Violaine Bonnefoy, Mark Dopson, and David S. Holmes. "Anaerobic Sulfur Metabolism Coupled to Dissimilatory Iron Reduction in the Extremophile Acidithiobacillus ferrooxidans." Applied and Environmental Microbiology 79, no. 7 (January 25, 2013): 2172–81. http://dx.doi.org/10.1128/aem.03057-12.
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ABSTRACTGene transcription (microarrays) and protein levels (proteomics) were compared in cultures of the acidophilic chemolithotrophAcidithiobacillus ferrooxidansgrown on elemental sulfur as the electron donor under aerobic and anaerobic conditions, using either molecular oxygen or ferric iron as the electron acceptor, respectively. No evidence supporting the role of either tetrathionate hydrolase or arsenic reductase in mediating the transfer of electrons to ferric iron (as suggested by previous studies) was obtained. In addition, no novel ferric iron reductase was identified. However, data suggested that sulfur was disproportionated under anaerobic conditions, forming hydrogen sulfide via sulfur reductase and sulfate via heterodisulfide reductase and ATP sulfurylase. Supporting physiological evidence for H2S production came from the observation that soluble Cu2+included in anaerobically incubated cultures was precipitated (seemingly as CuS). Since H2S reduces ferric iron to ferrous in acidic medium, its production under anaerobic conditions indicates that anaerobic iron reduction is mediated, at least in part, by an indirect mechanism. Evidence was obtained for an alternative model implicating the transfer of electrons from S0to Fe3+via a respiratory chain that includes abc1complex and a cytochromec. Central carbon pathways were upregulated under aerobic conditions, correlating with higher growth rates, while many Calvin-Benson-Bassham cycle components were upregulated during anaerobic growth, probably as a result of more limited access to carbon dioxide. These results are important for understanding the role ofA. ferrooxidansin environmental biogeochemical metal cycling and in industrial bioleaching operations.
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Xia, Chuanwu, Djemel Hamdane, Anna L. Shen, Vivian Choi, Charles B. Kasper, Naw May Pearl, Haoming Zhang, Sang-Choul Im, Lucy Waskell, and Jung-Ja P. Kim. "Conformational Changes of NADPH-Cytochrome P450 Oxidoreductase Are Essential for Catalysis and Cofactor Binding." Journal of Biological Chemistry 286, no. 18 (February 23, 2011): 16246–60. http://dx.doi.org/10.1074/jbc.m111.230532.
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The crystal structure of NADPH-cytochrome P450 reductase (CYPOR) implies that a large domain movement is essential for electron transfer from NADPH via FAD and FMN to its redox partners. To test this hypothesis, a disulfide bond was engineered between residues Asp147 and Arg514 in the FMN and FAD domains, respectively. The cross-linked form of this mutant protein, designated 147CC514, exhibited a significant decrease in the rate of interflavin electron transfer and large (≥90%) decreases in rates of electron transfer to its redox partners, cytochrome c and cytochrome P450 2B4. Reduction of the disulfide bond restored the ability of the mutant to reduce its redox partners, demonstrating that a conformational change is essential for CYPOR function. The crystal structures of the mutant without and with NADP+ revealed that the two flavin domains are joined by a disulfide linkage and that the relative orientations of the two flavin rings are twisted ∼20° compared with the wild type, decreasing the surface contact area between the two flavin rings. Comparison of the structures without and with NADP+ shows movement of the Gly631–Asn635 loop. In the NADP+-free structure, the loop adopts a conformation that sterically hinders NADP(H) binding. The structure with NADP+ shows movement of the Gly631–Asn635 loop to a position that permits NADP(H) binding. Furthermore, comparison of these mutant and wild type structures strongly suggests that the Gly631–Asn635 loop movement controls NADPH binding and NADP+ release; this loop movement in turn facilitates the flavin domain movement, allowing electron transfer from FMN to the CYPOR redox partners.
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Mayer, Sonja, Wojtek Steffen, Julia Steuber, and Friedrich Götz. "The Staphylococcus aureus NuoL-Like Protein MpsA Contributes to the Generation of Membrane Potential." Journal of Bacteriology 197, no. 5 (December 1, 2014): 794–806. http://dx.doi.org/10.1128/jb.02127-14.
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In aerobic microorganisms, the entry point of respiratory electron transfer is represented by the NADH:quinone oxidoreductase. The enzyme couples the oxidation of NADH with the reduction of quinone. In the type 1 NADH:quinone oxidoreductase (Ndh1), this reaction is accompanied by the translocation of cations, such as H+or Na+. InEscherichia coli, cation translocation is accomplished by the subunit NuoL, thus generating membrane potential (Δψ). Some microorganisms achieve NADH oxidation by the alternative, nonelectrogenic type 2 NADH:quinone oxidoreductase (Ndh2), which is not cation translocating. Since these enzymes had not been described inStaphylococcus aureus, the goal of this study was to identify proteins operating in the NADH:quinone segment of its respiratory chain. We demonstrated that Ndh2 represents a NADH:quinone oxidoreductase inS. aureus. Additionally, we identified a hypothetical protein inS. aureusshowing sequence similarity to the proton-translocating subunit NuoL of complex I inE. coli: the NuoL-like protein MpsA. Mutants with deletion of thenuoL-like genempsAand its corresponding operon,mpsABC(mpsfor membrane potential-generating system), exhibited a small-colony-variant-like phenotype and were severely affected in Δψ and oxygen consumption rates. The MpsABC proteins did not confer NADH oxidation activity. Using an Na+/H+antiporter-deficientE. colistrain, we could show that MpsABC constitute a cation-translocating system capable of Na+transport. Our study demonstrates that MpsABC represent an important functional system of the respiratory chain ofS. aureusthat acts as an electrogenic unit responsible for the generation of Δψ.
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Halestrap, A. P., and J. L. Dunlop. "Intramitochondrial regulation of fatty acid β-oxidation occurs between flavoprotein and ubiquinone A role for changes in the matrix volume." Biochemical Journal 239, no. 3 (November 1, 1986): 559–65. http://dx.doi.org/10.1042/bj2390559.
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Rat liver mitochondria were incubated in media of different osmolarities and in the presence of various substrates. Rates of oxygen consumption and mitochondrial matrix volumes were measured in the presence and absence of ADP and uncoupler. Duroquinol oxidation was insensitive to matrix volume, whereas other substrates tested showed increased rates of oxidation when the matrix volume increased from 1.0 to 1.5 microliter/mg of protein; this is the range of values measured in situ [Quinlan, Thomas, Armston & Halestrap (1983) Biochem. J. 214, 395-404]. Palmitoylcarnitine, octanoate and butyrate oxidations were particularly sensitive to the matrix volume, increasing from negligible rates to maximal rates within this range. Swelling induced by K+ uptake also stimulated palmitoylcarnitine oxidation. A similar effect of volume on substrate oxidation was seen when ferricyanide in the presence or absence of ubiquinone-1 replaced oxygen as terminal electron acceptor. Measurement of flavoprotein reduction (A 460-480) demonstrated that the locus of the effect of matrix volume is between the electron-transfer flavoprotein and ubiquinone. It is suggested that volume-mediated regulation of fatty acid and proline oxidation may be an important component of the hormonal stimulation of their oxidation.
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Taniguchi, Seiji, Haik Chosrowjan, Fumio Tanaka, Takeshi Nakanishi, Shuta Sato, Yoshihiro Haruyama, and Masaya Kitamura. "A Key Factor for Ultrafast Rates of Photoinduced Electron Transfer among Five Flavin Mononucleotide Binding Proteins: Effect of Negative, Positive, and Neutral Charges at Residue 13 on the Rate." Bulletin of the Chemical Society of Japan 86, no. 3 (March 15, 2013): 339–50. http://dx.doi.org/10.1246/bcsj.20120231.
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Fisher, K., D. J. Lowe, and R. N. F. Thorneley. "Klebsiella pneumoniae nitrogenase. The pre-steady-state kinetics of MoFe-protein reduction and hydrogen evolution under conditions of limiting electron flux show that the rates of association with the Fe-protein and electron transfer are independent of the oxidation level of the MoFe-protein." Biochemical Journal 279, no. 1 (October 1, 1991): 81–85. http://dx.doi.org/10.1042/bj2790081.
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The pre-steady-state kinetics of H2 evolution from Klebsiella pneumoniae nitrogenase functioning at 23 degrees C, pH 7.4, under conditions of extremely low electron flux through the MoFe-protein exhibited a lag phase of several minutes duration. The approach to a steady-state rate of H2 evolution was accompanied by a 50% decrease in the amplitude of the MoFe-protein e.p.r. signal. These kinetics have been simulated using our published kinetic model for nitrogenase [Lowe & Thorneley (1984) Biochem. J. 224, 877-886], which was developed using data obtained with nitrogenase functioning at high electron fluxes. The e.p.r. data showed that the rate of complex-formation between reduced Fe-protein and the MoFe-protein (k+1 = 5 x 10(7) M-1.s-1) is the same for the resting (E0) and one-electron-reduced (E1H) states of the MoFe-protein. Stopped-flow spectrophotometry also showed that electron transfer from the Fe-protein to the MoFe-protein in states E0 and E1H occurs at the same rate (kobs. = 140 s-1). These data support our previous assumption that the rate constants that define the ‘Fe-protein cycle’ are independent of the level of reduction of the MoFe-protein.
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Vasilev, Cvetelin, Guy E. Mayneord, Amanda A. Brindley, Matthew P. Johnson, and C. Neil Hunter. "Dissecting the cytochrome c2–reaction centre interaction in bacterial photosynthesis using single molecule force spectroscopy." Biochemical Journal 476, no. 15 (August 9, 2019): 2173–90. http://dx.doi.org/10.1042/bcj20170519.
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Abstract The reversible docking of small, diffusible redox proteins onto a membrane protein complex is a common feature of bacterial, mitochondrial and photosynthetic electron transfer (ET) chains. Spectroscopic studies of ensembles of such redox partners have been used to determine ET rates and dissociation constants. Here, we report a single-molecule analysis of the forces that stabilise transient ET complexes. We examined the interaction of two components of bacterial photosynthesis, cytochrome c2 and the reaction centre (RC) complex, using dynamic force spectroscopy and PeakForce quantitative nanomechanical imaging. RC–LH1–PufX complexes, attached to silicon nitride AFM probes and maintained in a photo-oxidised state, were lowered onto a silicon oxide substrate bearing dispersed, immobilised and reduced cytochrome c2 molecules. Microscale patterns of cytochrome c2 and the cyan fluorescent protein were used to validate the specificity of recognition between tip-attached RCs and surface-tethered cytochrome c2. Following the transient association of photo-oxidised RC and reduced cytochrome c2 molecules, retraction of the RC-functionalised probe met with resistance, and forces between 112 and 887 pN were required to disrupt the post-ET RC–c2 complex, depending on the retraction velocities used. If tip-attached RCs were reduced instead, the probability of interaction with reduced cytochrome c2 molecules decreased 5-fold. Thus, the redox states of the cytochrome c2 haem cofactor and RC ‘special pair’ bacteriochlorophyll dimer are important for establishing a productive ET complex. The millisecond persistence of the post-ET cytochrome c2[oxidised]–RC[reduced] ‘product’ state is compatible with rates of cyclic photosynthetic ET, at physiologically relevant light intensities.
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Pössl, Axel, David Hartzke, Peggy Schlupp, and Frank E. Runkel. "Calculation of Mass Transfer and Cell-Specific Consumption Rates to Improve Cell Viability in Bioink Tissue Constructs." Materials 14, no. 16 (August 5, 2021): 4387. http://dx.doi.org/10.3390/ma14164387.
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Biofabrication methods such as extrusion-based bioprinting allow the manufacture of cell-laden structures for cell therapy, but it is important to provide a sufficient number of embedded cells for the replacement of lost functional tissues. To address this issue, we investigated mass transfer rates across a bioink hydrogel for the essential nutrients glucose and glutamine, their metabolites lactate and ammonia, the electron acceptor oxygen, and the model protein bovine serum albumin. Diffusion coefficients were calculated for these substances at two temperatures. We could confirm that diffusion depends on the molecular volume of the substances if the bioink has a high content of polymers. The analysis of pancreatic 1.1B4 β-cells revealed that the nitrogen source glutamine is a limiting nutrient for homeostasis during cultivation. Taking the consumption rates of 1.1B4 β-cells into account during cultivation, we were able to calculate the cell numbers that can be adequately supplied by the cell culture medium and nutrients in the blood using a model tissue construct. For blood-like conditions, a maximum of ~106 cells·mL−1 was suitable for the cell-laden construct, as a function of the diffused substrate and cell consumption rate for a given geometry. We found that oxygen and glutamine were the limiting nutrients in our model.
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Davies, K. L., E. J. Camm, D. J. Smith, O. R. Vaughan, A. J. Forhead, A. J. Murray, and A. L. Fowden. "Glucocorticoid maturation of mitochondrial respiratory capacity in skeletal muscle before birth." Journal of Endocrinology 251, no. 1 (October 1, 2021): 53–68. http://dx.doi.org/10.1530/joe-21-0171.
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In adults, glucocorticoids act to match the supply and demand for energy during physiological challenges, partly through actions on tissue mitochondrial oxidative phosphorylation (OXPHOS) capacity. However, little is known about the role of the natural prepartum rise in fetal glucocorticoid concentrations in preparing tissues for the increased postnatal energy demands. This study examined the effect of manipulating cortisol concentrations in fetal sheep during late gestation on mitochondrial OXPHOS capacity of two skeletal muscles with different postnatal locomotive functions. Mitochondrial content, biogenesis markers, respiratory rates and expression of proteins and genes involved in the electron transfer system (ETS) and OXPHOS efficiency were measured in the biceps femoris (BF) and superficial digital flexor (SDF) of fetuses either infused with cortisol before the prepartum rise or adrenalectomised to prevent this increment. Cortisol infusion increased mitochondrial content, biogenesis markers, substrate-specific respiration rates and abundance of ETS complex I and adenine nucleotide translocator (ANT1) in a muscle-specific manner that was more pronounced in the SDF than BF. Adrenalectomy reduced mitochondrial content and expression of PGC1α and ANT1 in both muscles, and ETS complex IV abundance in the SDF near term. Uncoupling protein gene expression was unaffected by cortisol manipulations in both muscles. Gene expression of the myosin heavy chain isoform, MHCIIx, was increased by cortisol infusion and reduced by adrenalectomy in the BF alone. These findings show that cortisol has a muscle-specific role in prepartum maturation of mitochondrial OXPHOS capacity with important implications for the health of neonates born pre-term or after intrauterine glucocorticoid overexposure.
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Warshel, Arieh, and William W. Parson. "Dynamics of biochemical and biophysical reactions: insight from computer simulations." Quarterly Reviews of Biophysics 34, no. 4 (November 2001): 563–679. http://dx.doi.org/10.1017/s0033583501003730.
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1. Introduction 5632. Obtaining rate constants from molecular-dynamics simulations 5642.1 General relationships between quantum electronic structures and reaction rates 5642.2 The transition-state theory (TST) 5692.3 The transmission coefficient 5723. Simulating biological electron-transfer reactions 5753.1 Semi-classical surface-hopping and the Marcus equation 5753.2 Treating quantum mechanical nuclear tunneling by the dispersed-polaron/spin-boson method 5803.3 Density-matrix treatments 5833.4 Charge separation in photosynthetic bacterial reaction centers 5844. Light-induced photoisomerizations in rhodopsin and bacteriorhodopsin 5965. Energetics and dynamics of enzyme reactions 6145.1 The empirical-valence-bond treatment and free-energy perturbation methods 6145.2 Activation energies are decreased in enzymes relative to solution, often by electrostatic effects that stabilize the transition state 6205.3 Entropic effects in enzyme catalysis 6275.4 What is meant by dynamical contributions to catalysis? 6345.5 Transmission coefficients are similar for corresponding reactions in enzymes and water 6365.6 Non-equilibrium solvation effects contribute to catalysis mainly through Δg[Dagger], not the transmission coefficient 6415.7 Vibrationally assisted nuclear tunneling in enzyme catalysis 6485.8 Diffusive processes in enzyme reactions and transmembrane channels 6516. Concluding remarks 6587. Acknowledgements 6588. References 658Obtaining a detailed understanding of the dynamics of a biochemical reaction is a formidable challenge. Indeed, it might appear at first sight that reactions in proteins are too complex to analyze microscopically. At room temperature, even a relatively small protein can have as many as 1034 accessible conformational states (Dill, 1985). In many cases, however, we have detailed structural information about the active site of an enzyme, whereas such information is missing for corresponding chemical systems in solution. The atomic coordinates of the chromophore in bacteriorhodopsin, for example, are known to a resolution of 1–2 Å. In addition, experimental studies of biological processes such as photoisomerization and electron transfer have provided a wealth of detailed information that eventually may make some of these processes classical problems in chemical physics as well as biology.
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Thiemann, Jutta, and Wolfgang Barz. "Photoautotrophic Chenopodium rubrum Cell Suspension Cultures Resistant against Photosynthesis-Inhibiting Herbicides II. Physiological and Biochemical Properties." Zeitschrift für Naturforschung C 49, no. 11-12 (December 1, 1994): 791–801. http://dx.doi.org/10.1515/znc-1994-11-1215.
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Eight photoautotrophic cell cultures of Chenopodium rubrum, which are resistant against the photosystem II inhibitor metribuzin, were characterized for their growth parameters, chlorophyll content and photosynthetic capacity. Herbicide resistance of the eight lines results from different mutations in the D 1 protein of photosystem II, which is the target for different photosystem II inhibitors. In the presence of 10-5 ᴍ metribuzin the eight lines showed substantial growth reduction depending on the degree of resistance, and this effect is explained by a reduced electron transport in photosystem II. The impaired photosynthetic capacity of the green cells in the presence of high metribuzin concentrations, leads to compensation effects similar to shade accommodation of plants. Adaptation includes an increase of the chlorophyll content, a decrease of the chlorophyll a/b ratios as well as an increase of thylakoid stacking and cell number per unit fresh weight. In the absence of the herbicide photosynthetic electron transport is not impaired, as indicated by measurements of electron transfer rates in photosystem II and flash-induced reduction kinetics of P-700+. In summary the alterations of the D 1 protein of the eight cell lines do not result in a reduced electron transport in photosystem II.
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Paulson, A. F., P. D. Lampe, R. A. Meyer, E. TenBroek, M. M. Atkinson, T. F. Walseth, and R. G. Johnson. "Cyclic AMP and LDL trigger a rapid enhancement in gap junction assembly through a stimulation of connexin trafficking." Journal of Cell Science 113, no. 17 (September 1, 2000): 3037–49. http://dx.doi.org/10.1242/jcs.113.17.3037.
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Given the rapid turnover of connexin proteins, gap junction (GJ) assembly represents an important means of regulating the extent of GJ communication between cells. This report describes an increase in the level of GJ assembly within one hour following treatment with cAMP-elevating reagents or low density lipoprotein (LDL). Dye transfer methods and freeze-fracture with electron microscopy were used to assay junctional permeability and structure, respectively, subsequent to the dissociation, recovery and reaggregation of Novikoff hepatoma cells. Reaggregating cells in the presence of agents that increase cAMP levels (8-Br-cAMP, forskolin and IBMX) enhanced both dye transfer rates between cells and the extent of GJ formation 2- to 3-fold. These data and studies with the protein kinase A inhibitor, H-89, indicate that cAMP signaling plays a key role in enhanced assembly. The response to LDL parallels that to cAMP and relies on the activity of both adenylyl cyclase and protein kinase A. Immunoblot analysis revealed no change in the level of connexin43 (Cx43) or its phosphorylation states over a period of 2.5 hours. However, three agents (brefeldin A, monensin and nocodazole), that inhibit intracellular membrane trafficking by different mechanisms, all blocked the enhanced assembly of GJs when triggered by either elevated cAMP or exposure to LDL. Related studies, which employed trafficking inhibitors at different stages in GJ assembly, suggested that Cx43 trafficking during enhanced assembly is regulated, in part, by cell contact. Intracellular sources of Cx43 were characterized by colabeling for several markers of cytoplasmic membrane systems. We conclude that an increase in GJ assembly: (i) occurs rapidly in the presence of elevated cAMP or LDL, (ii) does not require an increase in Cx43 levels or major changes in Cx43 phosphorylation and (iii) is dependent upon the trafficking of Cx43 from intracellular storage sites.
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Attia, Hamadi, Michel Bennasar, Alain Lagaude, Bernard Hugodot, Jacques Rouviere, and Blas Tarodo De La Fuente. "Ultrafiltration with a microfiltration membrane of acid skimmed and fat-enriched milk coagula: hydrodynamic, microscopic and rheological approaches." Journal of Dairy Research 60, no. 2 (May 1993): 161–74. http://dx.doi.org/10.1017/s0022029900027485.
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SummaryThe effect of acidification method (microbiological with or without renneting, HCl addition) on mass transfer, fouling structure and the rheology of the retentate was studied in the ultrafiltration of skim milk coagula using a mineral microfiltration membrane. The increase in fouling with time appeared to determine permeate flow rates, which were higher in biological coagula, and the protein retention rates which were higher in chemical coagula. Fouling was investigated using scanning electron microscopy. The rheological study showed that at the same total solids, biological coagula were more viscous than chemical coagula. The initial coagula (total solids 97 g/kg) all displayed pseudoplastic behaviour at low shear velocities and Newtonian behaviour at high velocities. Ultrafiltration of fat-enriched milk coagulum to a dry weight corresponding to a soft cheese (total solids 334 g/kg; fat in total solids 60%) gave satisfactory permeate flow rates and protein retention rates. Performance was related to the composition of the product, the hydrodynamic parameters used and the resulting fouling. The rheological study showed that the initial coagulum behaved as a pseudoplastic body at low shear rate and for higher velocities as a Newtonian liquid. The concentrated retenate behaved as an ideal viscoplastic body (Bingham body).
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Howitt, Crispin A., Pacer K. Udall, and Wim F. J. Vermaas. "Type 2 NADH Dehydrogenases in the CyanobacteriumSynechocystis sp. Strain PCC 6803 Are Involved in Regulation Rather Than Respiration." Journal of Bacteriology 181, no. 13 (July 1, 1999): 3994–4003. http://dx.doi.org/10.1128/jb.181.13.3994-4003.1999.
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ABSTRACT Analysis of the genome of Synechocystis sp. strain PCC 6803 reveals three open reading frames (slr0851,slr1743, and sll1484) that may code for type 2 NAD(P)H dehydrogenases (NDH-2). The sequence similarity between the translated open reading frames and NDH-2s from other organisms is low, generally not exceeding 30% identity. However, NAD(P)H and flavin adenine dinucleotide binding motifs are conserved in all three putative NDH-2s in Synechocystis sp. strain PCC 6803. The three open reading frames were cloned, and deletion constructs were made for each. An expression construct containing one of the three open reading frames, slr1743, was able to functionally complement anEscherichia coli mutant lacking both NDH-1s and NDH-2s. Therefore, slr0851, slr1743, andsll1484 have been designated ndbA,ndbB, and ndbC, respectively. Strains that lacked one or more of the ndb genes were created in wild-type and photosystem (PS) I-less backgrounds. Deletion ofndb genes led to small changes in photoautotrophic growth rates and respiratory activities. Electron transfer rates into the plastoquinone pool in thylakoids in darkness were consistent with the presence of a small amount of NDH-2 activity in thylakoids. No difference was observed between wild-type and the Ndb-less strains in the banding patterns seen on native gels when stained for either NADH or NADPH dehydrogenase activity, indicating that the Ndb proteins do not accumulate to high levels. A striking phenotype of the PS I-less background strains lacking one or more of the NDH-2s is that they were able to grow at high light intensities that were lethal to the control strain but they retained normal PS II activity. We suggest that the Ndb proteins in Synechocystis sp. strain PCC 6803 are redox sensors and that they play a regulatory role responding to the redox state of the plastoquinone pool.
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Crooks, Daniel R., Nunziata Maio, Andrew N. Lane, Michal Jarnik, Richard M. Higashi, Ronald G. Haller, Ye Yang, Teresa W.-M. Fan, W. Marston Linehan, and Tracey A. Rouault. "Acute loss of iron–sulfur clusters results in metabolic reprogramming and generation of lipid droplets in mammalian cells." Journal of Biological Chemistry 293, no. 21 (March 9, 2018): 8297–311. http://dx.doi.org/10.1074/jbc.ra118.001885.
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Iron–sulfur (Fe-S) clusters are ancient cofactors in cells and participate in diverse biochemical functions, including electron transfer and enzymatic catalysis. Although cell lines derived from individuals carrying mutations in the Fe-S cluster biogenesis pathway or siRNA-mediated knockdown of the Fe-S assembly components provide excellent models for investigating Fe-S cluster formation in mammalian cells, these experimental strategies focus on the consequences of prolonged impairment of Fe-S assembly. Here, we constructed and expressed dominant–negative variants of the primary Fe-S biogenesis scaffold protein iron–sulfur cluster assembly enzyme 2 (ISCU2) in human HEK293 cells. This approach enabled us to study the early metabolic reprogramming associated with loss of Fe-S–containing proteins in several major cellular compartments. Using multiple metabolomics platforms, we observed a ∼12-fold increase in intracellular citrate content in Fe-S–deficient cells, a surge that was due to loss of aconitase activity. The excess citrate was generated from glucose-derived acetyl-CoA, and global analysis of cellular lipids revealed that fatty acid biosynthesis increased markedly relative to cellular proliferation rates in Fe-S–deficient cells. We also observed intracellular lipid droplet accumulation in both acutely Fe-S–deficient cells and iron-starved cells. We conclude that deficient Fe-S biogenesis and acute iron deficiency rapidly increase cellular citrate concentrations, leading to fatty acid synthesis and cytosolic lipid droplet formation. Our findings uncover a potential cause of cellular steatosis in nonadipose tissues.