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1.
J Biol Chem ; 296: 100107, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33219127

RESUMO

A key step in bacteriochlorophyll biosynthesis is the reduction of protochlorophyllide to chlorophyllide, catalyzed by dark-operative protochlorophyllide oxidoreductase. Dark-operative protochlorophyllide oxidoreductase contains two [4Fe-4S]-containing component proteins (BchL and BchNB) that assemble upon ATP binding to BchL to coordinate electron transfer and protochlorophyllide reduction. But the precise nature of the ATP-induced conformational changes is poorly understood. We present a crystal structure of BchL in the nucleotide-free form where a conserved, flexible region in the N-terminus masks the [4Fe-4S] cluster at the docking interface between BchL and BchNB. Amino acid substitutions in this region produce a hyperactive enzyme complex, suggesting a role for the N-terminus in autoinhibition. Hydrogen-deuterium exchange mass spectrometry shows that ATP binding to BchL produces specific conformational changes leading to release of the flexible N-terminus from the docking interface. The release also promotes changes within the local environment surrounding the [4Fe-4S] cluster and promotes BchL-complex formation with BchNB. A key patch of amino acids, Asp-Phe-Asp (the 'DFD patch'), situated at the mouth of the BchL ATP-binding pocket promotes intersubunit cross stabilization of the two subunits. A linked BchL dimer with one defective ATP-binding site does not support protochlorophyllide reduction, illustrating nucleotide binding to both subunits as a prerequisite for the intersubunit cross stabilization. The masking of the [4Fe-4S] cluster by the flexible N-terminal region and the associated inhibition of the activity is a novel mechanism of regulation in metalloproteins. Such mechanisms are possibly an adaptation to the anaerobic nature of eubacterial cells with poor tolerance for oxygen.


Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Trifosfato de Adenosina/química , Catálise , Proteínas Ferro-Enxofre/química , Espectrometria de Massas , Nitrogenase/química , Nitrogenase/metabolismo , Fotossíntese , Protoclorifilida/química , Protoclorifilida/metabolismo , Especificidade por Substrato
2.
Am J Physiol Lung Cell Mol Physiol ; 320(3): L356-L367, 2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33325804

RESUMO

Idiopathic pulmonary fibrosis (IPF) is characterized by a disturbed redox balance and increased production of reactive oxygen species (ROS), which is believed to contribute to epithelial injury and fibrotic lung scarring. The main pulmonary sources of ROS include mitochondria and NADPH oxidases (NOXs), of which the NOX4 isoform has been implicated in IPF. Non-receptor SRC tyrosine kinases (SFK) are important for cellular homeostasis and are often dysregulated in lung diseases. SFK activation by the profibrotic transforming growth factor-ß (TGF-ß) is thought to contribute to pulmonary fibrosis, but the relevant SFK isoform and its relationship to NOX4 and/or mitochondrial ROS in the context of profibrotic TGF-ß signaling is not known. Here, we demonstrate that TGF-ß1 can rapidly activate the SRC kinase FYN in human bronchial epithelial cells, which subsequently induces mitochondrial ROS (mtROS) production, genetic damage shown by the DNA damage marker γH2AX, and increased expression of profibrotic genes. Moreover, TGF-ß1-induced activation of FYN involves initial activation of NOX4 and direct cysteine oxidation of FYN, and both FYN and mtROS contribute to TGF-ß-induced induction of NOX4. NOX4 expression in lung tissues of IPF patients is positively correlated with disease severity, although FYN expression is down-regulated in IPF and does not correlate with disease severity. Collectively, our findings highlight a critical role for FYN in TGF-ß1-induced mtROS production, DNA damage response, and induction of profibrotic genes in bronchial epithelial cells, and suggest that altered expression and activation of NOX4 and FYN may contribute to the pathogenesis of pulmonary fibrosis.


Assuntos
Brônquios/metabolismo , Células Epiteliais/metabolismo , Mitocôndrias/metabolismo , NADPH Oxidase 4/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta1/metabolismo , Brônquios/patologia , Células Epiteliais/patologia , Humanos , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Mitocôndrias/patologia , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
3.
Am J Respir Cell Mol Biol ; 63(2): 198-208, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32182090

RESUMO

The airway epithelium plays a critical role in innate responses to airborne allergens by secreting IL-1 family cytokines such as IL-1α and IL-33 as alarmins that subsequently orchestrate appropriate immune responses. Previous studies revealed that epithelial IL-33 secretion by allergens such as Alternaria alternata or house dust mite involves Ca2+-dependent signaling, via initial activation of ATP-stimulated P2YR2 (type 2 purinoceptor) and subsequent activation of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase DUOX1. We sought to identify proximal mechanisms by which epithelial cells sense these allergens and here highlight the importance of PAR2 (protease-activated receptor 2) and TRP (transient receptor potential) Ca2+ channels such as TRPV1 (TRP vanilloid 1) in these responses. Combined studies of primary human nasal and mouse tracheal epithelial cells, as well as immortalized human bronchial epithelial cells, indicated the importance of both PAR2 and TRPV1 in IL-33 secretion by both Alternaria alternata and house dust mite, based on both pharmacological and genetic approaches. TRPV1 was also critically involved in allergen-induced ATP release, activation of DUOX1, and redox-dependent activation of EGFR (epidermal growth factor receptor). Moreover, genetic deletion of TRPV1 dramatically attenuated allergen-induced IL-33 secretion and subsequent type 2 responses in mice in vivo. TRPV1 not only contributed to ATP release and P2YR2 signaling but also was critical in downstream innate responses to ATP, indicating potentiating effects of P2YR2 on TRPV1 activation. In aggregate, our studies illustrate a complex relationship between various receptor types, including PAR2 and P2YR2, in epithelial responses to asthma-relevant airborne allergens and highlight the central importance of TRPV1 in such responses.


Assuntos
Alérgenos/imunologia , Células Epiteliais/imunologia , Imunidade Inata/imunologia , Peptídeo Hidrolases/imunologia , Canais de Cátion TRPV/imunologia , Animais , Asma/imunologia , Brônquios/imunologia , Células Cultivadas , Epitélio/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Pyroglyphidae/imunologia , Receptor PAR-2/imunologia , Mucosa Respiratória/imunologia , Transdução de Sinais/imunologia
4.
J Biol Chem ; 293(25): 9629-9635, 2018 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-29720402

RESUMO

Nitrogenase is the enzyme that reduces atmospheric dinitrogen (N2) to ammonia (NH3) in biological systems. It catalyzes a series of single-electron transfers from the donor iron protein (Fe protein) to the molybdenum-iron protein (MoFe protein) that contains the iron-molybdenum cofactor (FeMo-co) sites where N2 is reduced to NH3 The P-cluster in the MoFe protein functions in nitrogenase catalysis as an intermediate electron carrier between the external electron donor, the Fe protein, and the FeMo-co sites of the MoFe protein. Previous work has revealed that the P-cluster undergoes redox-dependent structural changes and that the transition from the all-ferrous resting (PN) state to the two-electron oxidized P2+ state is accompanied by protein serine hydroxyl and backbone amide ligation to iron. In this work, the MoFe protein was poised at defined potentials with redox mediators in an electrochemical cell, and the three distinct structural states of the P-cluster (P2+, P1+, and PN) were characterized by X-ray crystallography and confirmed by computational analysis. These analyses revealed that the three oxidation states differ in coordination, implicating that the P1+ state retains the serine hydroxyl coordination but lacks the backbone amide coordination observed in the P2+ states. These results provide a complete picture of the redox-dependent ligand rearrangements of the three P-cluster redox states.


Assuntos
Azotobacter vinelandii/enzimologia , Molibdoferredoxina/química , Nitrogenase/química , Conformação Proteica , Prótons , Catálise , Cristalografia por Raios X , Transporte de Elétrons , Molibdoferredoxina/metabolismo , Nitrogenase/metabolismo , Oxirredução
5.
J Cell Biochem ; 120(8): 13783-13791, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30938854

RESUMO

The chimeric DnaJ-PKAc enzymeresulting from an approximately 400-kb deletion of chromosome 19 is a primary contributor to the oncogenic transformation that occurs in fibrolamellar hepatocellular carcinoma, also called fibrolamellar carcinoma (FLC). This oncogenic deletion juxtaposes exon 1 of the DNAJB1 heat shock protein gene with exon 2 of the PRKACA gene encoding the protein kinase A catalytic subunit, resulting in DnaJ-PKAc fusion under the transcriptional control of the DNAJB1 promoter. The expression of DnaJ-PKAc is approximately 10 times that of wild-type (wt) PKAc catalytic subunits, causing elevated and dysregulated kinase activity that contributes to oncogenic transformation. In normal cells, PKAc activity is regulated by a group of endogenous proteins, termed protein kinase inhibitors (PKI) that competitively inhibit PKAc and assist with the nuclear export of the enzyme. Currently, it is scarcely known whether interactions with PKI are perturbed in DnaJ-PKAc. In this report, we survey existing data sets to assess the expression levels of the various PKI isoforms that exist in humans to identify those that are candidates to encounter DnaJ-PKAc in both normal liver and FLC tumors. We then compare inhibition profiles of wtPKAc and DnaJ-PKAc against PKI and demonstrate that extensive structural homology in the active site clefts of the two enzymes confers similar kinase activities and inhibition by full-length PKI and PKI-derived peptides.


Assuntos
Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico , Proteínas de Choque Térmico HSP40 , Proteínas de Fusão Oncogênica , Peptídeos/química , Inibidores de Proteínas Quinases/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/antagonistas & inibidores , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/química , Subunidades Catalíticas da Proteína Quinase Dependente de AMP Cíclico/genética , Proteínas de Choque Térmico HSP40/antagonistas & inibidores , Proteínas de Choque Térmico HSP40/química , Proteínas de Choque Térmico HSP40/genética , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Proteínas de Fusão Oncogênica/antagonistas & inibidores , Proteínas de Fusão Oncogênica/química , Proteínas de Fusão Oncogênica/genética
6.
Proc Natl Acad Sci U S A ; 113(40): E5783-E5791, 2016 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-27698129

RESUMO

Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen (N2) to two ammonia (NH3) molecules through the participation of its two protein components, the MoFe and Fe proteins. Electron transfer (ET) from the Fe protein to the catalytic MoFe protein involves a series of synchronized events requiring the transient association of one Fe protein with each αß half of the α2ß2 MoFe protein. This process is referred to as the Fe protein cycle and includes binding of two ATP to an Fe protein, association of an Fe protein with the MoFe protein, ET from the Fe protein to the MoFe protein, hydrolysis of the two ATP to two ADP and two Pi for each ET, Pi release, and dissociation of oxidized Fe protein-(ADP)2 from the MoFe protein. Because the MoFe protein tetramer has two separate αß active units, it participates in two distinct Fe protein cycles. Quantitative kinetic measurements of ET, ATP hydrolysis, and Pi release during the presteady-state phase of electron delivery demonstrate that the two halves of the ternary complex between the MoFe protein and two reduced Fe protein-(ATP)2 do not undergo the Fe protein cycle independently. Instead, the data are globally fit with a two-branch negative-cooperativity kinetic model in which ET in one-half of the complex partially suppresses this process in the other. A possible mechanism for communication between the two halves of the nitrogenase complex is suggested by normal-mode calculations showing correlated and anticorrelated motions between the two halves.


Assuntos
Trifosfato de Adenosina/química , Molibdoferredoxina/química , Complexos Multiproteicos/química , Oxirredutases/química , Trifosfato de Adenosina/metabolismo , Animais , Transporte de Elétrons , Hidrólise , Cinética , Molibdoferredoxina/metabolismo , Complexos Multiproteicos/metabolismo , Fixação de Nitrogênio , Oxirredutases/metabolismo , Ligação Proteica , Salmão/metabolismo
7.
J Biol Chem ; 291(44): 23282-23293, 2016 10 28.
Artigo em Inglês | MEDLINE | ID: mdl-27650496

RESUMO

The epidermal growth factor receptor (EGFR) plays a critical role in regulating airway epithelial homeostasis and responses to injury. Activation of EGFR is regulated by redox-dependent processes involving reversible cysteine oxidation by reactive oxygen species (ROS) and involves both ligand-dependent and -independent mechanisms, but the precise source(s) of ROS and the molecular mechanisms that control tyrosine kinase activity are incompletely understood. Here, we demonstrate that stimulation of EGFR activation by ATP in airway epithelial cells is closely associated with dynamic reversible oxidation of cysteine residues via sequential sulfenylation and S-glutathionylation within EGFR and the non-receptor-tyrosine kinase Src. Moreover, the intrinsic kinase activity of recombinant Src or EGFR was in both cases enhanced by H2O2 but not by GSSG, indicating that the intermediate sulfenylation is the activating modification. H2O2-induced increase in EGFR tyrosine kinase activity was not observed with the C797S variant, confirming Cys-797 as the redox-sensitive cysteine residue that regulates kinase activity. Redox-dependent regulation of EGFR activation in airway epithelial cells was found to strongly depend on activation of either the NADPH oxidase DUOX1 or the homolog NOX2, depending on the activation mechanism. Whereas DUOX1 and Src play a primary role in EGFR transactivation by wound-derived signals such as ATP, direct ligand-dependent EGFR activation primarily involves NOX2 with a secondary role for DUOX1 and Src. Collectively, our findings establish that redox-dependent EGFR kinase activation involves a dynamic and reversible cysteine oxidation mechanism and that this activation mechanism variably involves DUOX1 and NOX2.


Assuntos
Receptores ErbB/metabolismo , Glicoproteínas de Membrana/metabolismo , NADPH Oxidases/metabolismo , Transdução de Sinais , Animais , Oxidases Duais , Células Epiteliais/enzimologia , Células Epiteliais/metabolismo , Receptores ErbB/genética , Feminino , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , NADPH Oxidase 2 , NADPH Oxidases/genética , Oxirredução , Espécies Reativas de Oxigênio/metabolismo
8.
Am J Physiol Lung Cell Mol Physiol ; 311(5): L913-L923, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27612966

RESUMO

Acrolein is a major thiol-reactive component of cigarette smoke (CS) that is thought to contribute to increased asthma incidence associated with smoking. Here, we explored the effects of acute acrolein exposure on innate airway responses to two common airborne allergens, house dust mite and Alternaria alternata, and observed that acrolein exposure of C57BL/6 mice (5 ppm, 4 h) dramatically inhibited innate airway responses to subsequent allergen challenge, demonstrated by attenuated release of the epithelial-derived cytokines IL-33, IL-25, and IL-1α. Acrolein and other anti-inflammatory thiol-reactive electrophiles, cinnamaldehyde, curcumin, and sulforaphane, similarly inhibited allergen-induced production of these cytokines from human or murine airway epithelial cells in vitro. Based on our previous observations indicating the importance of Ca2+-dependent signaling, activation of the NADPH oxidase DUOX1, and Src/EGFR-dependent signaling in allergen-induced epithelial secretion of these cytokines, we explored the impact of acrolein on these pathways. Acrolein and other thiol-reactive electrophiles were found to dramatically prevent allergen-induced activation of DUOX1 as well as EGFR, and acrolein was capable of inhibiting EGFR tyrosine kinase activity via modification of C797. Biotin-labeling strategies indicated increased cysteine modification and carbonylation of Src, EGFR, as well as DUOX1, in response to acrolein exposure in vitro and in vivo, suggesting that direct alkylation of these proteins on accessible cysteine residues may be responsible for their inhibition. Collectively, our findings indicate a novel anti-inflammatory mechanism of CS-derived acrolein and other thiol-reactive electrophiles, by directly inhibiting DUOX1- and EGFR-mediated airway epithelial responses to airborne allergens.


Assuntos
Acroleína/farmacologia , Alérgenos/efeitos adversos , Brônquios/patologia , Células Epiteliais/metabolismo , Receptores ErbB/metabolismo , NADPH Oxidases/antagonistas & inibidores , Compostos de Sulfidrila/farmacologia , Acroleína/química , Administração por Inalação , Animais , Cálcio/metabolismo , Cisteína/metabolismo , Oxidases Duais , Ativação Enzimática/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Humanos , Peróxido de Hidrogênio/metabolismo , Imunidade Inata/efeitos dos fármacos , Interleucina-33/metabolismo , Camundongos Endogâmicos C57BL , NADPH Oxidases/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Pyroglyphidae/efeitos dos fármacos , Pyroglyphidae/fisiologia , Compostos de Sulfidrila/química , Quinases da Família src/metabolismo
9.
Proc Natl Acad Sci U S A ; 110(41): 16414-9, 2013 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-24062462

RESUMO

The biological reduction of N2 to NH3 catalyzed by Mo-dependent nitrogenase requires at least eight rounds of a complex cycle of events associated with ATP-driven electron transfer (ET) from the Fe protein to the catalytic MoFe protein, with each ET coupled to the hydrolysis of two ATP molecules. Although steps within this cycle have been studied for decades, the nature of the coupling between ATP hydrolysis and ET, in particular the order of ET and ATP hydrolysis, has been elusive. Here, we have measured first-order rate constants for each key step in the reaction sequence, including direct measurement of the ATP hydrolysis rate constant: kATP = 70 s(-1), 25 °C. Comparison of the rate constants establishes that the reaction sequence involves four sequential steps: (i) conformationally gated ET (kET = 140 s(-1), 25 °C), (ii) ATP hydrolysis (kATP = 70 s(-1), 25 °C), (iii) Phosphate release (kPi = 16 s(-1), 25 °C), and (iv) Fe protein dissociation from the MoFe protein (kdiss = 6 s(-1), 25 °C). These findings allow completion of the thermodynamic cycle undergone by the Fe protein, showing that the energy of ATP binding and protein-protein association drive ET, with subsequent ATP hydrolysis and Pi release causing dissociation of the complex between the Fe(ox)(ADP)2 protein and the reduced MoFe protein.


Assuntos
Trifosfato de Adenosina/metabolismo , Azotobacter vinelandii/metabolismo , Modelos Biológicos , Fixação de Nitrogênio/fisiologia , Oxirredutases/metabolismo , Transporte de Elétrons/fisiologia , Hidrólise , Cinética , Espectrofotometria , Termodinâmica
10.
Biochemistry ; 54(15): 2456-62, 2015 Apr 21.
Artigo em Inglês | MEDLINE | ID: mdl-25831270

RESUMO

The reduction of substrates catalyzed by nitrogenase normally requires nucleotide-dependent Fe protein delivery of electrons to the MoFe protein, which contains the active site FeMo cofactor. Here, it is reported that independent substitution of three amino acids (ß-98(Tyr→His), α-64(Tyr→His), and ß-99(Phe→His)) located between the P cluster and FeMo cofactor within the MoFe protein endows it with the ability to reduce protons to H2, azide to ammonia, and hydrazine to ammonia without the need for Fe protein or ATP. Instead, electrons can be provided by the low-potential reductant polyaminocarboxylate-ligated Eu(II) (Em values of -1.1 to -0.84 V vs the normal hydrogen electrode). The crystal structure of the ß-98(Tyr→His) variant MoFe protein was determined, revealing only small changes near the amino acid substitution that affect the solvent structure and the immediate vicinity between the P cluster and the FeMo cofactor, with no global conformational changes observed. Computational normal-mode analysis of the nitrogenase complex reveals coupling in the motions of the Fe protein and the region of the MoFe protein with these three amino acids, which suggests a possible mechanism for how Fe protein might communicate subtle changes deep within the MoFe protein that profoundly affect intramolecular electron transfer and substrate reduction.


Assuntos
Azotobacter vinelandii/enzimologia , Proteínas de Bactérias/química , Coenzimas/química , Simulação por Computador , Ferro/química , Molibdênio/química , Nitrogenase/química , Trifosfato de Adenosina/química , Substituição de Aminoácidos , Azotobacter vinelandii/genética , Proteínas de Bactérias/genética , Coenzimas/genética , Mutação de Sentido Incorreto , Nitrogenase/genética
11.
Biochemistry ; 53(14): 2278-85, 2014 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-24654842

RESUMO

Mo-dependent nitrogenase catalyzes the biological reduction of N2 to two NH3 molecules at FeMo-cofactor buried deep inside the MoFe protein. Access of substrates, such as N2, to the active site is likely restricted by the surrounding protein, requiring substrate channels that lead from the surface to the active site. Earlier studies on crystallographic structures of the MoFe protein have suggested three putative substrate channels. Here, we have utilized submicrosecond atomistic molecular dynamics simulations to allow the nitrogenase MoFe protein to explore its conformational space in an aqueous solution at physiological ionic strength, revealing a putative substrate channel. The viability of this observed channel was tested by examining the free energy of passage of N2 from the surface through the channel to FeMo-cofactor, resulting in the discovery of a very low energy barrier. These studies point to a viable substrate channel in nitrogenase that appears during thermal motions of the protein in an aqueous environment and that approaches a face of FeMo-cofactor earlier implicated in substrate binding.


Assuntos
Simulação de Dinâmica Molecular , Nitrogenase/química , Especificidade por Substrato
12.
J Am Chem Soc ; 136(36): 12776-83, 2014 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-25136926

RESUMO

Investigations of reduction of nitrite (NO2(-)) to ammonia (NH3) by nitrogenase indicate a limiting stoichiometry, NO2(-) + 6e(-) + 12ATP + 7H(+) → NH3 + 2H2O + 12ADP + 12Pi. Two intermediates freeze-trapped during NO2(-) turnover by nitrogenase variants and investigated by Q-band ENDOR/ESEEM are identical to states, denoted H and I, formed on the pathway of N2 reduction. The proposed NO2(-) reduction intermediate hydroxylamine (NH2OH) is a nitrogenase substrate for which the H and I reduction intermediates also can be trapped. Viewing N2 and NO2(-) reductions in light of their common reduction intermediates and of NO2(-) reduction by multiheme cytochrome c nitrite reductase (ccNIR) leads us to propose that NO2(-) reduction by nitrogenase begins with the generation of NO2H bound to a state in which the active-site FeMo-co (M) has accumulated two [e(-)/H(+)] (E2), stored as a (bridging) hydride and proton. Proton transfer to NO2H and H2O loss leaves M-[NO(+)]; transfer of the E2 hydride to the [NO(+)] directly to form HNO bound to FeMo-co is one of two alternative means for avoiding formation of a terminal M-[NO] thermodynamic "sink". The N2 and NO2(-) reduction pathways converge upon reduction of NH2NH2 and NH2OH bound states to form state H with [-NH2] bound to M. Final reduction converts H to I, with NH3 bound to M. The results presented here, combined with the parallels with ccNIR, support a N2 fixation mechanism in which liberation of the first NH3 occurs upon delivery of five [e(-)/H(+)] to N2, but a total of seven [e(-)/H(+)] to FeMo-co when obligate H2 evolution is considered, and not earlier in the reduction process.


Assuntos
Hidroxilamina/metabolismo , Nitritos/metabolismo , Nitrogênio/metabolismo , Nitrogenase/metabolismo , Hidroxilamina/química , Nitritos/química , Nitrogênio/química , Nitrogenase/química , Oxirredução , Especificidade por Substrato
13.
Inorg Chem ; 53(7): 3688-93, 2014 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-24635454

RESUMO

We have advanced a mechanism for nitrogenase catalysis that rests on the identification of a low-spin EPR signal (S = 1/2) trapped during turnover of a MoFe protein as the E4 state, which has accumulated four reducing equivalents as two [Fe-H-Fe] bridging hydrides. Because electrons are delivered to the MoFe protein one at a time, with the rate-limiting step being the off-rate of oxidized Fe protein, it is difficult to directly control, or know, the degree of reduction, n, of a trapped intermediate, denoted En, n = 1-8. To overcome this previously intractable problem, we introduced a quench-cryoannealing relaxation protocol for determining n of an EPR-active trapped En turnover state. The trapped "hydride" state was allowed to relax to the resting E0 state in frozen medium, which prevents additional accumulation of reducing equivalents; binding of reduced Fe protein and release of oxidized protein from the MoFe protein both are abolished in a frozen solid. Relaxation of En was monitored by periodic EPR analysis at cryogenic temperature. The protocol rests on the hypothesis that an intermediate trapped in the frozen solid can relax toward the resting state only by the release of a stable reduction product from FeMo-co. In turnover under Ar, the only product that can be released is H2, which carries two reducing equivalents. This hypothesis implicitly predicts that states that have accumulated an odd number of electrons/protons (n = 1, 3) during turnover under Ar cannot relax to E0: E3 can relax to E1, but E1 cannot relax to E0 in the frozen state. The present experiments confirm this prediction and, thus, the quench-cryoannealing protocol and our assignment of E4, the foundation of the proposed mechanism for nitrogenase catalysis. This study further gives insights into the identity of the En intermediates with high-spin EPR signals, 1b and 1c, trapped under high electron flux.


Assuntos
Nitrogenase/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Congelamento , Ferro/química , Ferro/metabolismo , Molibdênio/química , Molibdênio/metabolismo , Molibdoferredoxina/metabolismo , Nitrogenase/química , Oxirredução
14.
J Bacteriol ; 195(2): 279-86, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23144248

RESUMO

Fe protein (dinitrogenase reductase) activity is reversibly inactivated by dinitrogenase reductase ADP-ribosyltransferase (DraT) in response to an increase in the ammonium concentration or a decrease in cellular energy in Azospirillum brasilense, Rhodospirillum rubrum, and Rhodobacter capsulatus. The ADP-ribosyl is removed by the dinitrogenase reductase-activating glycohydrolase (DraG), promoting Fe protein reactivation. The signaling pathway leading to DraT activation by ammonium is still not completely understood, but the available evidence shows the involvement of direct interaction between the enzyme and the nitrogen-signaling P(II) proteins. In A. brasilense, two P(II) proteins, GlnB and GlnZ, were identified. We used Fe protein from Azotobacter vinelandii as the substrate to assess the activity of A. brasilense DraT in vitro complexed or not with P(II) proteins. Under our conditions, GlnB was necessary for DraT activity in the presence of Mg-ADP. The P(II) effector 2-oxoglutarate, in the presence of Mg-ATP, inhibited DraT-GlnB activity, possibly by inducing complex dissociation. DraT was also activated by GlnZ and by both uridylylated P(II) proteins, but not by a GlnB variant carrying a partial deletion of the T loop. Kinetics studies revealed that the A. brasilense DraT-GlnB complex was at least 18-fold more efficient than DraT purified from R. rubrum, but with a similar K(m) value for NAD(+). Our results showed that ADP-ribosylation of the Fe protein does not affect the electronic state of its metal cluster and prevents association between the Fe and MoFe proteins, thus inhibiting electron transfer.


Assuntos
ADP Ribose Transferases/metabolismo , Azospirillum brasilense/enzimologia , Proteínas de Bactérias/metabolismo , Proteínas PII Reguladoras de Nitrogênio/metabolismo , ADP Ribose Transferases/isolamento & purificação , Difosfato de Adenosina/metabolismo , Azotobacter vinelandii/enzimologia , Coenzimas , Inibidores Enzimáticos/metabolismo , Ácidos Cetoglutáricos/metabolismo , Cinética , Magnésio/metabolismo , NAD/metabolismo , Oxirredutases/isolamento & purificação , Oxirredutases/metabolismo , Ligação Proteica
15.
Biochemistry ; 51(42): 8391-8, 2012 10 23.
Artigo em Inglês | MEDLINE | ID: mdl-23050654

RESUMO

Earlier studies of electron transfer (ET) from the nitrogenase Fe protein to the MoFe protein concluded that the mechanism for ET changed during cooling from 25 to 5 °C, based on the observation that the rate constant for Fe protein to MoFe protein ET decreases strongly, with a nonlinear Arrhenius plot. They further indicated that the ET was reversible, with complete ET at ambient temperature but with an equilibrium constant near unity at 5 °C. These studies were conducted with buffers having a strong temperature coefficient. We have examined the temperature variation in the kinetics of oxidation of the Fe protein by the MoFe protein at a constant pH of 7.4 fixed by the buffer 3-(N-morpholino)propanesulfonic acid (MOPS), which has a very small temperature coefficient. Using MOPS, we also observe temperature-dependent ET rate constants, with nonlinear Arrhenius plots, but we find that ET is gated across the temperature range by a conformational change that involves the binding of numerous water molecules, consistent with an unchanging ET mechanism. Furthermore, there is no solvent kinetic isotope effect throughout the temperature range studied, again consistent with an unchanging mechanism. In addition, the nonlinear Arrhenius plots are explained by the change in heat capacity caused by the binding of waters in an invariant gating ET mechanism. Together, these observations contradict the idea of a change in ET mechanism with cooling. Finally, the extent of ET at constant pH does not change significantly with temperature, in contrast to the previously proposed change in ET equilibrium.


Assuntos
Transporte de Elétrons , Nitrogenase/química , Nitrogenase/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Molibdoferredoxina/química , Morfolinas/química , Conformação Proteica , Temperatura
16.
Biochemistry ; 50(43): 9255-63, 2011 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-21939270

RESUMO

The reduction of substrates catalyzed by nitrogenase utilizes an electron transfer (ET) chain comprised of three metalloclusters distributed between the two component proteins, designated as the Fe protein and the MoFe protein. The flow of electrons through these three metalloclusters involves ET from the [4Fe-4S] cluster located within the Fe protein to an [8Fe-7S] cluster, called the P cluster, located within the MoFe protein and ET from the P cluster to the active site [7Fe-9S-X-Mo-homocitrate] cluster called FeMo-cofactor, also located within the MoFe protein. The order of these two electron transfer events, the relevant oxidation states of the P-cluster, and the role(s) of ATP, which is obligatory for ET, remain unknown. In the present work, the electron transfer process was examined by stopped-flow spectrophotometry using the wild-type MoFe protein and two variant MoFe proteins, one having the ß-188(Ser) residue substituted by cysteine and the other having the ß-153(Cys) residue deleted. The data support a "deficit-spending" model of electron transfer where the first event (rate constant 168 s(-1)) is ET from the P cluster to FeMo-cofactor and the second, "backfill", event is fast ET (rate constant >1700 s(-1)) from the Fe protein [4Fe-4S] cluster to the oxidized P cluster. Changes in osmotic pressure reveal that the first electron transfer is conformationally gated, whereas the second is not. The data for the ß-153(Cys) deletion MoFe protein variant provide an argument against an alternative two-step "hopping" ET model that reverses the two ET steps, with the Fe protein first transferring an electron to the P cluster, which in turn transfers an electron to FeMo-cofactor. The roles for ATP binding and hydrolysis in controlling the ET reactions were examined using ßγ-methylene-ATP as a prehydrolysis ATP analogue and ADP + AlF(4)(-) as a posthydrolysis analogue (a mimic of ADP + P(i)).


Assuntos
Azotobacter vinelandii/enzimologia , Nitrogenase/química , Nitrogenase/metabolismo , Azotobacter vinelandii/química , Transporte de Elétrons , Elétrons , Modelos Moleculares , Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Nucleotídeos/química , Nucleotídeos/metabolismo
17.
J Assoc Genet Technol ; 47(3): 127-131, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34491231

RESUMO

OBJECTIVES: Atypical chronic myeloid leukemia, BCR-ABL1-negative (aCML), is a rare myelodysplastic/myeloproliferative neoplasm with heterogeneous clinical and genetic features, a high rate of transformation to acute myeloid leukemia (AML), and poor survival rate. The diagnosis of aCML is a diagnosis of exclusion and requires the fulfillment of strict diagnostic criteria. Until recently, there were no distinctive cytogenetic or molecular abnormalities for aCML adding to the diagnostic challenge. We present a case of aCML and highlight the pertinent clinical, morphological, and genetic features required for the diagnosis.

18.
J Am Chem Soc ; 132(20): 6894-5, 2010 May 26.
Artigo em Inglês | MEDLINE | ID: mdl-20429505

RESUMO

The nitrogenase Fe protein contains a [4Fe-4S] cluster and delivers one electron at a time to the catalytic MoFe protein. During this electron delivery, the Fe protein in its [4Fe-4S](1+) reduced state (Fe(red)) binds two MgATP and forms a complex with the MoFe protein, with subsequent transfer of one electron to the MoFe protein in a reaction coupled to the hydrolysis of two ATP. Crystal structures with the nitrogenase complex in different nucleotide-bound states show major conformational changes which provide a structural underpinning to suggestions that intercomponent electron transfer (ET) is "gated" by conformational changes of the complex and/or of its component proteins. Although electron delivery is coupled to ATP hydrolysis, their connection is puzzling, for it appears that ET precedes both ATP hydrolysis and Pi release. We here test the gating hypothesis with studies of the intracomplex oxidation of Fe(red) by MoFe protein in the presence of a variety of solutes. Conformational control of this process (gating) is revealed by the finding that it responds to changes in osmotic pressure (but not viscosity), with no fewer than 80 waters being bound during the reaction. The absence of a solvent kinetic isotope effect further implies that ATP hydrolysis does not occur during the rate-limiting step of ET.


Assuntos
Molibdoferredoxina/química , Molibdoferredoxina/metabolismo , Oxirredutases/química , Oxirredutases/metabolismo , Trifosfato de Adenosina/metabolismo , Transporte de Elétrons , Hidrólise , Cinética , Conformação Proteica
19.
J Am Chem Soc ; 132(38): 13197-9, 2010 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-20812745

RESUMO

The catalytic reduction of hydrazine (N(2)H(4)) to ammonia by a ß-98(Tyr→His) MoFe protein in the absence of the Fe protein or ATP is reported. The reduction of N(2) or other substrates (e.g., hydrazine, protons, acetylene) by nitrogenase normally requires the transient association of the two nitrogenase component proteins, the Fe protein and the MoFe protein. The Fe protein, with two bound MgATP molecules, transfers one electron to the MoFe protein during each association, coupled to the hydrolysis of two MgATP. All substrate reduction reactions catalyzed by nitrogenase require delivery of electrons by the Fe protein coupled to the hydrolysis of MgATP. We report that when a single amino acid within the MoFe protein (ß-98(Tyr)) is substituted by His, the resulting MoFe protein supports catalytic reduction of the nitrogenous substrate hydrazine (N(2)H(4)) to two ammonia molecules when provided with a low potential reductant, polyaminocarboxylate ligated Eu(II) (E(m) -1.1 V vs NHE). The wild-type and a number of other MoFe proteins with amino acid substitutions do not show significant rates of hydrazine reduction under these conditions, whereas the ß-98(His) MoFe protein catalyzes hydrazine reduction at rates up to 170 nmol NH(3)/min/mg MoFe protein. This rate of hydrazine reduction is 94% of the rate catalyzed by the ß-98(His) or wild-type MoFe protein when combined with the Fe protein, ATP, and reductant under comparable conditions. The ß-98(His) MoFe protein reduction of hydrazine in the absence of the Fe protein showed saturation kinetics for the concentration of reductant and substrate. The implications of these results in understanding the nitrogenase mechanism are discussed.


Assuntos
Trifosfato de Adenosina/química , Amônia/química , Hidrazinas/química , Ferro/química , Molibdênio/química , Proteínas/química , Catálise , Modelos Moleculares
20.
Sci Rep ; 9(1): 4844, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30890751

RESUMO

Lung cancers are frequently characterized by inappropriate activation of epidermal growth factor receptor (EGFR)-dependent signaling and epigenetic silencing of the NADPH oxidase (NOX) enzyme DUOX1, both potentially contributing to worse prognosis. Based on previous findings linking DUOX1 with redox-dependent EGFR activation, the present studies were designed to evaluate whether DUOX1 silencing in lung cancers may be responsible for altered EGFR regulation. In contrast to normal epithelial cells, EGF stimulation of lung cancer cell lines that lack DUOX1 promotes EGF-induced EGFR internalization and nuclear localization, associated with induction of EGFR-regulated genes and related tumorigenic outcomes. Each of these outcomes could be reversed by overexpression of DUOX1 or enhanced by shRNA-dependent DUOX1 silencing. EGF-induced nuclear EGFR localization in DUOX1-deficient lung cancer cells was associated with altered dynamics of cysteine oxidation of EGFR, and an overall reduction of EGFR cysteines. These various outcomes could also be attenuated by silencing of glutathione S-transferase P1 (GSTP1), a mediator of metabolic alterations and drug resistance in various cancers, and a regulator of cysteine oxidation. Collectively, our findings indicate DUOX1 deficiency in lung cancers promotes dysregulated EGFR signaling and enhanced GSTP1-mediated turnover of EGFR cysteine oxidation, which result in enhanced nuclear EGFR localization and tumorigenic properties.


Assuntos
Nucléolo Celular/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Células A549 , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Oxidases Duais/metabolismo , Receptores ErbB/metabolismo , Humanos , NADPH Oxidases/metabolismo , Oxirredução , Transdução de Sinais/fisiologia
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