scholarly journals Electron Transfer and Binding of thec-Type Cytochrome TorC to the TrimethylamineN-Oxide Reductase inEscherichia coli

2000 ◽  
Vol 276 (15) ◽  
pp. 11545-11551 ◽  
Author(s):  
Stéphanie Gon ◽  
Marie-Thérèse Giudici-Orticoni ◽  
Vincent Méjean ◽  
Chantal Iobbi-Nivol
Keyword(s):  
Electron Transfer ◽  
Biochemical Characterization ◽  
Reduced Form ◽  
Visible Absorption ◽  
Midpoint Potential ◽  
Redox Potentiometry ◽  
Family Based ◽  
Transfer Chain

Reduction of trimethylamineN-oxide (E′0(TMAO/TMA)= +130 mV) inEscherichia coliis carried out by the Tor system, an electron transfer chain encoded by thetorCADoperon and made up of the periplasmic terminal reductase TorA and the membrane-anchored pentahemicc-type cytochrome TorC. Although the role of TorA in the reduction of trimethylamineN-oxide (TMAO) has been clearly established, no direct evidence for TorC involvement has been presented. TorC belongs to the NirT/NapCc-type cytochrome family based on homologies of its N-terminal tetrahemic domain (TorCN) to the cytochromes of this family, but TorC contains a C-terminal extension (TorCC) with an additional heme-binding site. In this study, we show that both domains are required for the anaerobic bacterial growth with TMAO. The intact TorC protein and its two domains, TorCNand TorCC, were produced independently and purified for a biochemical characterization. The reduced form of TorC exhibited visible absorption maxima at 552, 523, and 417 nm. Mediated redox potentiometry of the heme centers of the purified components identified two negative midpoint potentials (−177 and −98 mV) localized in the tetrahemic TorCNand one positive midpoint potential (+120 mV) in the monohemic TorCC. In agreement with these values, thein vitroreconstitution of electron transfer between TorC, TorCN, or TorCCand TorA showed that only TorC and TorCCwere capable of electron transfer to TorA. Surprisingly, interaction studies revealed that only TorC and TorCNstrongly bind TorA. Therefore, TorCCdirectly transfers electrons to TorA, whereas TorCN, which probably receives electrons from the menaquinone pool, is involved in both the electron transfer to TorCCand the binding to TorA.

scholarly journals A thylakoid membrane-bound and redox-active rubredoxin (RBD1) functions in de novo assembly and repair of photosystem II

2019 ◽  
Vol 116 (33) ◽  
pp. 16631-16640 ◽  
Author(s):  
José G. García-Cerdán ◽  
Ariel L. Furst ◽  
Kent L. McDonald ◽  
Danja Schünemann ◽  
Matthew B. Francis ◽  
...  
Keyword(s):  
Photosystem Ii ◽  
Thylakoid Membrane ◽  
De Novo Assembly ◽  
Biochemical Characterization ◽  
De Novo ◽  
Midpoint Potential ◽  
Photosynthetic Organisms ◽  
Redox Active ◽  
Photooxidative Damage

Photosystem II (PSII) undergoes frequent photooxidative damage that, if not repaired, impairs photosynthetic activity and growth. How photosynthetic organisms protect vulnerable PSII intermediate complexes during de novo assembly and repair remains poorly understood. Here, we report the genetic and biochemical characterization of chloroplast-located rubredoxin 1 (RBD1), a PSII assembly factor containing a redox-active rubredoxin domain and a single C-terminal transmembrane α-helix (TMH) domain. RBD1 is an integral thylakoid membrane protein that is enriched in stroma lamellae fractions with the rubredoxin domain exposed on the stromal side. RBD1 also interacts with PSII intermediate complexes containing cytochrome b559. Complementation of the Chlamydomonas reinhardtii (hereafter Chlamydomonas) RBD1-deficient 2pac mutant with constructs encoding RBD1 protein truncations and site-directed mutations demonstrated that the TMH domain is essential for de novo PSII assembly, whereas the rubredoxin domain is involved in PSII repair. The rubredoxin domain exhibits a redox midpoint potential of +114 mV and is proficient in 1-electron transfers to a surrogate cytochrome c in vitro. Reduction of oxidized RBD1 is NADPH dependent and can be mediated by ferredoxin-NADP+ reductase (FNR) in vitro. We propose that RBD1 participates, together with the cytochrome b559, in the protection of PSII intermediate complexes from photooxidative damage during de novo assembly and repair. This role of RBD1 is consistent with its evolutionary conservation among photosynthetic organisms and the fact that it is essential in photosynthetic eukaryotes.

scholarly journals Functional genetic encoding of sulfotyrosine in mammalian cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinyuan He ◽  
Yan Chen ◽  
Daisy Guiza Beltran ◽  
Maia Kelly ◽  
Bin Ma ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Trna Synthetase ◽  
Functional Verification ◽  
Cellular Processes ◽  
Genetic Encoding ◽  
Efficient Expression

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

Biochemical characterization of human DNA polymerase i provides clues to its biological function

2001 ◽  
Vol 29 (2) ◽  
pp. 183-187 ◽  
Author(s):  
A. Tissier ◽  
E. G. Frank ◽  
J. P. McDonald ◽  
A. Vaisman ◽  
A. R. Fernàndez deHenestrosa Henestrosa ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Biochemical Characterization ◽  
Short Review ◽  
Biochemical Properties ◽  
Dna Polymerase I ◽  
Polymerase I

The human RAD30B gene has recently been shown to encode a novel DNA polymerase, DNA polymerase i (poli). The role of poli within the cell is presently unknown, and the only clues to its cellular function come from its biochemical characterization in vitro. The aim of this short review is, therefore, to summarize the known enzymic activities of poli and to speculate as to how these biochemical properties might relate to its in vivo function.

Role of the Middle Residue in the Triple Tryptophan Electron Transfer Chain of DNA Photolyase:  Ultrafast Spectroscopy of a Trp→Phe Mutant

2006 ◽  
Vol 110 (32) ◽  
pp. 15654-15658 ◽  
Author(s):  
Andras Lukacs ◽  
André P. M. Eker ◽  
Martin Byrdin ◽  
Sandrine Villette ◽  
Jie Pan ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Ultrafast Spectroscopy ◽  
Dna Photolyase ◽  
Electron Transfer Chain ◽  
Transfer Chain

scholarly journals Disrupting Mitochondrial Electron Transfer Chain Complex I Decreases Immune Checkpoints in Murine and Human Acute Myeloid Leukemic Cells

2021 ◽  
Author(s):  
Raquel Luna-Yolba ◽  
Justine Marmoiton ◽  
Véronique Gigo ◽  
Xavier Marechal ◽  
Emeline Boet ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Cell Lines ◽  
Chain Complex ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Immune Checkpoints ◽  
Electron Transfer Chain ◽  
Transfer Chain ◽  
Acute Myeloid

Abstract: Oxidative metabolism is crucial for leukemic stem cell (LSC) function and drug resistance in acute myeloid leukemia (AML). Mitochondrial metabolism also affects the immune system and therefore the antitumor response. Modulation of oxidative phosphorylation (OxPHOS) has emerged as a promising approach to improve therapy outcome for AML patients. However, the effect of mitochondrial inhibitors on the immune compartment in the context of AML is yet to be explored. Immune checkpoints such as the ecto-nucleotidase CD39 and programmed dead ligand 1 (PD-L1) have been reported to be expressed in AML and linked to chemoresistance and poor prognosis. In the present study, we first demonstrated that a novel selective electron transfer chain complex (ETC) I inhibitor, EVT-701, decreased OxPHOS metabolism of murine and human cytarabine (AraC)-resistant leukemic cell lines. Furthermore, we showed that, while AraC induced immune response regulation by increasing CD39 expression and by reinforcing interferon-γ/PD-L1 axis, EVT-701 reduced CD39 and PD-L1 expression in vitro in a panel of both murine and human AML cell lines, especially upon AraC treatment. Altogether, this work uncovers a non-canonical function of ETCI in controlling CD39 and PD-L1 immune checkpoints, thereby improving the anti-tumor response in AML.

scholarly journals Photoactivation of Cryptochromes Invokes Competing Inter- and Intramolecular Electron Transfer

2020 ◽  
Author(s):  
Ruslan N. Tazhigulov ◽  
Justin Provazza ◽  
David Coker ◽  
Ksenia B. Bravaya
Keyword(s):  
Electron Transfer ◽  
Photoinduced Electron Transfer ◽  
Migratory Birds ◽  
Intramolecular Electron Transfer ◽  
Tryptophan Residues ◽  
Theoretical Evidence ◽  
Excited Charge Transfer State

<div>Growing experimental and theoretical evidence points to the key role of cryptochrome proteins in magnetoreception by migratory birds and insects. Cryptochrome photoactivation is achieved through a cascade of electron transfer events leading to formation of a long-lived spin-correlated radical pair. The electron transfer cascade is initiated by photoexcitation of the FAD cofactor and subsequent electron transfer through three conserved tryptophan residues, the so-called tryptophan triad. Presence of ATP was shown to increase the yield of the semireduced form of FAD. While electron transfer through the tryptophan triad is well characterized by both theoretical and experimental methods, the effects of ATP binding are still not well understood. The present work aims to unravel the mechanism of ultrafast photoinduced electron transfer in a cryptochrome protein with a focus on effects of ATP on the FAD photoreduction process. Photoinduced electron transfer is described by means of state-of-the-art theoretical methods: a hybrid quantum-classical polarizable embedding scheme is utilized to accurately parameterize a generalized local excited/charge transfer state system-bath model Hamiltonian and the photoinduced electron transfer process is described by a semiclassical path integral-based dynamics method. The results draw attention to the crucial role of the intramolecular electron transfer from adenine to the flavin moiety of the FAD cofactor for formation of the semireduced form of FAD, providing an explanation for the increased yield of the semireduced form in the presence of the cellular metabolites <i>in vitro</i> and <i>in vivo</i>.</div>

Sign in / Sign up

Export Citation Format

Share Document