Distinct electron transfer from ferredoxin–thioredoxin reductase to multiple thioredoxin isoforms in chloroplasts

2017 ◽  
Vol 474 (8) ◽  
pp. 1347-1360 ◽  
Author(s):  
Keisuke Yoshida ◽  
Toru Hisabori
Keyword(s):  
Electron Transfer ◽  
Redox Regulation ◽  
Thioredoxin Reductase ◽  
Reducing Power ◽  
Redox Potentials ◽  
Regulation Network ◽  
Electron Transfers ◽  
Kinetics Of

Thiol-based redox regulation is considered to support light-responsive control of various chloroplast functions. The redox cascade via ferredoxin–thioredoxin reductase (FTR)/thioredoxin (Trx) has been recognized as a key to transmitting reducing power; however, Arabidopsis thaliana genome sequencing has revealed that as many as five Trx subtypes encoded by a total of 10 nuclear genes are targeted to chloroplasts. Because each Trx isoform seems to have a distinct target selectivity, the electron distribution from FTR to multiple Trxs is thought to be the critical branch point for determining the consequence of chloroplast redox regulation. In the present study, we aimed to comprehensively characterize the kinetics of electron transfer from FTR to 10 Trx isoforms. We prepared the recombinant FTR protein from Arabidopsis in the heterodimeric form containing the Fe–S cluster. By reconstituting the FTR/Trx system in vitro, we showed that FTR prepared here was enzymatically active and suitable for uncovering biochemical features of chloroplast redox regulation. A series of redox state determinations using the thiol-modifying reagent, 4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate, indicated that all chloroplast Trx isoforms are commonly reduced by FTR; however, significantly different efficiencies were evident. These differences were apparently correlated with the distinct midpoint redox potentials among Trxs. Even when the experiments were performed under conditions of hypothetical in vivo stoichiometry of FTR and Trxs, a similar trend in distinguishable electron transfers was observed. These data highlight an aspect of highly organized circuits in the chloroplast redox regulation network.

scholarly journals Thioredoxin-like2/2-Cys peroxiredoxin redox cascade supports oxidative thiol modulation in chloroplasts

2018 ◽  
Vol 115 (35) ◽  
pp. E8296-E8304 ◽  
Author(s):  
Keisuke Yoshida ◽  
Ayaka Hara ◽  
Kazunori Sugiura ◽  
Yuki Fukaya ◽  
Toru Hisabori
Keyword(s):  
Redox Regulation ◽  
High Efficiency ◽  
Thioredoxin Reductase ◽  
Reducing Power ◽  
Delayed Response ◽  
Physiological Relevance ◽  
Direct Discrimination ◽  
Chloroplast Stroma

Thiol-based redox regulation is central to adjusting chloroplast functions under varying light conditions. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been well recognized to mediate the light-responsive reductive control of target proteins; however, the molecular basis for reoxidizing its targets in the dark remains unidentified. Here, we report a mechanism of oxidative thiol modulation in chloroplasts. We biochemically characterized a chloroplast stroma-localized atypical Trx from Arabidopsis, designated as Trx-like2 (TrxL2). TrxL2 had redox-active properties with an unusually less negative redox potential. By an affinity chromatography-based method, TrxL2 was shown to interact with a range of chloroplast redox-regulated proteins. The direct discrimination of thiol status indicated that TrxL2 can efficiently oxidize, but not reduce, these proteins. A notable exception was found in 2-Cys peroxiredoxin (2CP); TrxL2 was able to reduce 2CP with high efficiency. We achieved a complete in vitro reconstitution of the TrxL2/2CP redox cascade for oxidizing redox-regulated proteins and draining reducing power to hydrogen peroxide (H2O2). We further addressed the physiological relevance of this system by analyzing protein-oxidation dynamics. In Arabidopsis plants, a decreased level of 2CP led to the impairment of the reoxidation of redox-regulated proteins during light–dark transitions. A delayed response of protein reoxidation was concomitant with the prolonged accumulation of reducing power in TrxL2. These results suggest an in vivo function of the TrxL2/2CP redox cascade for driving oxidative thiol modulation in chloroplasts.

scholarly journals Determining the Rate-Limiting Step for Light-Responsive Redox Regulation in Chloroplasts

Antioxidants ◽  
2018 ◽  
Vol 7 (11) ◽  
pp. 153 ◽  
Author(s):  
Keisuke Yoshida ◽  
Toru Hisabori
Keyword(s):  
Redox Regulation ◽  
Reducing Power ◽  
Rubisco Activase ◽  
Power Transfer ◽  
Target Proteins ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Rate Limiting

Thiol-based redox regulation ensures light-responsive control of chloroplast functions. Light-derived signal is transferred in the form of reducing power from the photosynthetic electron transport chain to several redox-sensitive target proteins. Two types of protein, ferredoxin-thioredoxin reductase (FTR) and thioredoxin (Trx), are well recognized as the mediators of reducing power. However, it remains unclear which step in a series of redox-relay reactions is the critical bottleneck for determining the rate of target protein reduction. To address this, the redox behaviors of FTR, Trx, and target proteins were extensively characterized in vitro and in vivo. The FTR/Trx redox cascade was reconstituted in vitro using recombinant proteins from Arabidopsis. On the basis of this assay, we found that the FTR catalytic subunit and f-type Trx are rapidly reduced after the drive of reducing power transfer, irrespective of the presence or absence of their downstream target proteins. By contrast, three target proteins, fructose 1,6-bisphosphatase (FBPase), sedoheptulose 1,7-bisphosphatase (SBPase), and Rubisco activase (RCA) showed different reduction patterns; in particular, SBPase was reduced at a low rate. The in vivo study using Arabidopsis plants showed that the Trx family is commonly and rapidly reduced upon high light irradiation, whereas FBPase, SBPase, and RCA are differentially and slowly reduced. Both of these biochemical and physiological findings suggest that reducing power transfer from Trx to its target proteins is a rate-limiting step for chloroplast redox regulation, conferring distinct light-responsive redox behaviors on each of the targets.

scholarly journals Mutations of the Membrane-Bound Disulfide Reductase DsbD That Block Electron Transfer Steps from Cytoplasm to Periplasm in Escherichia coli

2006 ◽  
Vol 188 (14) ◽  
pp. 5066-5076 ◽  
Author(s):  
Seung-Hyun Cho ◽  
Jon Beckwith
Keyword(s):  
Electron Transfer ◽  
Amino Acid ◽  
Transfer Process ◽  
Reducing Power ◽  
Amino Acid Residues ◽  
Electron Transfer Process ◽  
Redox States ◽  
Clear Cut

ABSTRACT The cytoplasmic membrane protein DsbD keeps the periplasmic disulfide isomerase DsbC reduced, using the cytoplasmic reducing power of thioredoxin. DsbD contains three domains, each containing two reactive cysteines. One membrane-embedded domain, DsbDβ, transfers electrons from thioredoxin to the carboxy-terminal thioredoxin-like periplasmic domain DsbDγ. To evaluate the role of conserved amino acid residues in DsbDβ in the electron transfer process, we substituted alanines for each of 19 conserved amino acid residues and assessed the in vivo redox states of DsbC and DsbD. The mutant DsbDs of 11 mutants which caused defects in DsbC reduction showed relatively oxidized redox states. To analyze the redox state of each DsbD domain, we constructed a thrombin-cleavable DsbD (DsbDTH) from which we could generate all three domains as separate polypeptide chains by thrombin treatment in vitro. We divided the mutants with strong defects into two classes. The first mutant class consists of mutant DsbDβ proteins that cannot receive electrons from cytoplasmic thioredoxin, resulting in a DsbD that has all six of its cysteines disulfide bonded. The second mutant class represents proteins in which the transfer of electrons from DsbDβ to DsbDγ appears to be blocked. This class includes the mutant with the most clear-cut defect, P284A. We relate the properties of the mutants to the positions of the amino acids in the structure of DsbD and discuss mechanisms that would interfere with the electron transfer process.

scholarly journals Staphylococcus aureus NrdH Redoxin Is a Reductant of the Class Ib Ribonucleotide Reductase

2010 ◽  
Vol 192 (19) ◽  
pp. 4963-4972 ◽  
Author(s):  
Inbal Rabinovitch ◽  
Michaela Yanku ◽  
Adva Yeheskel ◽  
Gerald Cohen ◽  
Ilya Borovok ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Ribonucleotide Reductase ◽  
Thioredoxin Reductase ◽  
Reducing Power ◽  
Anaerobic Growth ◽  
Structural Motif ◽  
Thiol Redox ◽  
Class Ib

ABSTRACT Staphylococci contain a class Ib NrdEF ribonucleotide reductase (RNR) that is responsible, under aerobic conditions, for the synthesis of deoxyribonucleotide precursors for DNA synthesis and repair. The genes encoding that RNR are contained in an operon consisting of three genes, nrdIEF, whereas many other class Ib RNR operons contain a fourth gene, nrdH, that determines a thiol redoxin protein, NrdH. We identified a 77-amino-acid open reading frame in Staphylococcus aureus that resembles NrdH proteins. However, S. aureus NrdH differs significantly from the canonical NrdH both in its redox-active site, C-P-P-C instead of C-M/V-Q-C, and in the absence of the C-terminal [WF]SGFRP[DE] structural motif. We show that S. aureus NrdH is a thiol redox protein. It is not essential for aerobic or anaerobic growth and appears to have a marginal role in protection against oxidative stress. In vitro, S. aureus NrdH was found to be an efficient reductant of disulfide bonds in low-molecular-weight substrates and proteins using dithiothreitol as the source of reducing power and an effective reductant for the homologous class Ib RNR employing thioredoxin reductase and NADPH as the source of the reducing power. Its ability to reduce NrdEF is comparable to that of thioredoxin-thioredoxin reductase. Hence, S. aureus contains two alternative thiol redox proteins, NrdH and thioredoxin, with both proteins being able to function in vitro with thioredoxin reductase as the immediate hydrogen donors for the class Ib RNR. It remains to be clarified under which in vivo physiological conditions the two systems are used.

scholarly journals Mammalian thioredoxin reductase 1: roles in redox homoeostasis and characterization of cellular targets

2010 ◽  
Vol 430 (2) ◽  
pp. 285-293 ◽  
Author(s):  
Anton A. Turanov ◽  
Sebastian Kehr ◽  
Stefano M. Marino ◽  
Min-Hyuk Yoo ◽  
Bradley A. Carlson ◽  
...  
Keyword(s):  
Redox Regulation ◽  
Thioredoxin Reductase ◽  
In Vitro Assays ◽  
Mouse Serum ◽  
Main Function ◽  
Major Pathway ◽  
Thioredoxin System

The classical Trx (thioredoxin) system, composed of TR (Trx reductase), Trx and NADPH, defines a major pathway of cellular thiol-based redox regulation. Three TRs have been identified in mammals: (i) cytosolic TR1, (ii) mitochondrial TR3 and (iii) testes-specific TGR (Trx-glutathione reductase). All three are selenocysteine-containing enzymes with broad substrate specificity in in vitro assays, but which protein substrates are targeted by TRs in vivo is not well understood. In the present study, we used a mechanism-based approach to characterize the molecular targets of TR1. Cytosolic Trx1 was the major target identified in rat and mouse liver, as well as in rat brain and mouse serum. The results suggest that the main function of TR1 is to reduce Trx1. We also found that TR1-based affinity resins provide a convenient tool for specific isolation of Trxs from a variety of biological samples. To better assess the role of TRs in redox homoeostasis, we comparatively analysed TR1- and TR3-knockdown cells. Although cells deficient in TR1 were particularly sensitive to diamide, TR3-knockdown cells were more sensitive to hydrogen peroxide. To further examine the TR1–Trx1 redox pair, we used mice with a liver-specific knockout of selenocysteine tRNA. In this model, selenocysteine insertion into TR1 was blocked, but the truncated form of this protein was not detected. Instead, TR1 and TR3 levels were decreased in the knockout samples. Diminished hepatic TR1 function was associated with elevated Trx1 levels, but this protein was mostly in the oxidized state. Overall, this study provides evidence for the key role of the TR1–Trx1 pair in redox homoeostasis.

KINETICS OF PHOTOCHEMICAL ELECTRON TRANSFER REACTIONS IN VIVO AND IN VITRO

1978 ◽  
pp. 37-44 ◽  
Author(s):  
W.W. Parson ◽  
C.C. Schenck ◽  
R.E. Blankenship ◽  
D. Holten ◽  
M.W. Windsor ◽  
...  
Keyword(s):  
Electron Transfer ◽  
Transfer Reactions ◽  
Electron Transfer Reactions ◽  
Kinetics Of

scholarly journals Oxidative regulation of chloroplast enzymes by thioredoxin and thioredoxin-like proteins in Arabidopsis thaliana

2021 ◽  
Vol 118 (51) ◽  
pp. e2114952118
Author(s):  
Yuichi Yokochi ◽  
Yuka Fukushi ◽  
Ken-ichi Wakabayashi ◽  
Keisuke Yoshida ◽  
Toru Hisabori
Keyword(s):  
Redox Regulation ◽  
Reducing Power ◽  
Photosynthetic Electron Transport ◽  
Rubisco Activase ◽  
Thermal Dissipation ◽  
Fructose 1,6 Bisphosphatase ◽  
Related Proteins ◽  
Oxidative Regulation

Thioredoxin (Trx) is a protein that mediates the reducing power transfer from the photosynthetic electron transport system to target enzymes in chloroplasts and regulates their activities. Redox regulation governed by Trx is a system that is central to the adaptation of various chloroplast functions to the ever-changing light environment. However, the factors involved in the opposite reaction (i.e., the oxidation of various enzymes) have yet to be revealed. Recently, it has been suggested that Trx and Trx-like proteins could oxidize Trx-targeted proteins in vitro. To elucidate the in vivo function of these proteins as oxidation factors, we generated mutant plant lines deficient in Trx or Trx-like proteins and studied how the proteins are involved in oxidative regulation in chloroplasts. We found that f-type Trx and two types of Trx-like proteins, Trx-like 2 and atypical Cys His-rich Trx (ACHT), seemed to serve as oxidation factors for Trx-targeted proteins, such as fructose-1,6-bisphosphatase, Rubisco activase, and the γ-subunit of ATP synthase. In addition, ACHT was found to be involved in regulating nonphotochemical quenching, which is the mechanism underlying the thermal dissipation of excess light energy. Overall, these results indicate that Trx and Trx-like proteins regulate chloroplast functions in concert by controlling the redox state of various photosynthesis-related proteins in vivo.

Effect of pH and inhibitors on beta-amyloid fibril assembly

1996 ◽  
Vol 54 ◽  
pp. 752-753
Author(s):  
Beverly E. Maleeff ◽  
Timothy K. Hart ◽  
Stephen J. Wood ◽  
Ronald Wetzel
Keyword(s):  
Amyloid Fibril ◽  
Peptide Fragment ◽  
Hydrophobic Peptides ◽  
Amyloid Fibril Formation ◽  
Effects Of Ph ◽  
Severity Of The Disease ◽  
Kinetics Of ◽  
The Brain

Alzheimer's disease is characterized post-mortem in part by abnormal extracellular neuritic plaques found in brain tissue. There appears to be a correlation between the severity of Alzheimer's dementia in vivo and the number of plaques found in particular areas of the brain. These plaques are known to be the deposition sites of fibrils of the protein β-amyloid. It is thought that if the assembly of these plaques could be inhibited, the severity of the disease would be decreased. The peptide fragment Aβ, a precursor of the p-amyloid protein, has a 40 amino acid sequence, and has been shown to be toxic to neuronal cells in culture after an aging process of several days. This toxicity corresponds to the kinetics of in vitro amyloid fibril formation. In this study, we report the biochemical and ultrastructural effects of pH and the inhibitory agent hexadecyl-N-methylpiperidinium (HMP) bromide, one of a class of ionic micellar detergents known to be capable of solubilizing hydrophobic peptides, on the in vitro assembly of the peptide fragment Aβ.

Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

1977 ◽  
Vol 16 (04) ◽  
pp. 157-162 ◽  
Author(s):  
C. Schümichen ◽  
B. Mackenbrock ◽  
G. Hoffmann
Keyword(s):  
Normal Saline ◽  
Half Life ◽  
Compound A ◽  
Short Half Life ◽  
Low Concentrations ◽  
The Stability ◽  
Kinetics Of ◽  
In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

Acquired Haemophilia: Functional Study of Antibodies to Factor VIII

1981 ◽  
Vol 45 (03) ◽  
pp. 285-289 ◽  
Author(s):  
J P Allain ◽  
A Gaillandre ◽  
D Frommel
Keyword(s):  
Factor Viii ◽  
Functional Study ◽  
Factor Viii Inhibitor ◽  
Acquired Haemophilia ◽  
Viii Inhibitor ◽  
Kinetics Of ◽  
Antibody Function ◽  
Native Plasma

SummaryFactor VIII complex and its interaction with antibodies to factor VIII have been studied in 17 non-haemophilic patients with factor VIII inhibitor. Low VIII:C and high VIIIR.Ag levels were found in all patients. VIII:WF levels were 50% of those of VTIIRrAg, possibly related to an increase of poorly aggregated and electrophoretically fast moving VIIIR:Ag oligomers.Antibody function has been characterized by kinetics of VIII :C inactivation, saturability by normal plasma and the slope of the affinity curve. Two major patterns were observed:1) Antibodies from 6 patients behaved similarly to those from haemophiliacs by showing second order inhibition kinetics, easy saturability and steep affinity slope (> 1).2) Antibodies from other patients, usually with lower titres, inactivated VIII :C according to complex order kinetics, were not saturable, and had a less steep affinity slope (< 0.7). In native plasma, or after mixing with factor VIII concentrate, antibodies of the second group did not form immune complexes with the whole factor VIII molecular complex. However, dissociation procedures did release some antibodies from apparently low molecular weight complexes formed in vivo or in vitro. For appropriate management of non-haemophilic patients with factor VIII inhibitor, it is important to determine the functional properties of their antibodies to factor VIII.

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