Molecular Dynamics Simulation for Probing the Flexibility of the 35 Nucleotide SL1 Sequence Kissing Complex from HIV-1Lai Genomic RNA

Abstract

The SL1 stem-loop located in the encapsidation domain is responsible for initiating the dimerisation of HIV-1 genomic RNA by means of a loop-loop interaction known as Kissing Complex (KC). The SL1 secondary structure has been predicted as a 35 nucleotides [K. G. Murti, M. Bondurant, and A. Tereba. J Virol 37, 411–419 (1981)] stem-loop composed of a 4 base pairs (bp) terminal duplex, a 4 nt asymmetrical internal loop, a 7 bp internal duplex, and a 9 nt apical loop. Several high resolution structures of the monomer and of KC of a 23 nt sequence containing only the internal duplex and the apical loop of SL1 are available in the literature. No experimental high resolution structure of the complete native SL1 sequence has been reported so far, either for the monomer or for KC. The asymmetrical internal loop has been described from NMR studies of different monomeric hairpin sequences, leading to divergent results, which suggests its high flexibility. In this work, we built a SL1₃₅ KC model which was submitted to a 31 ns molecular dynamics simulation (MD).

Our results allows to describe the internal dynamics of SL1₃₅ KC and the differences of behavior of the different parts of the dimer. Thus, we could show the stability of the interactions between the two apical loops and of the terminal duplexes, the destabilization of the internal duplexes and the high flexibility of the asymmetrical internal loops.     

Molecular Dynamics and Free Energy Calculations of the B and Z Forms of C8-Arylguanine Modified Oligonucleotides

Abstract

Arylhydrazines found in the mushroom Agaricus bisporus have been shown to be carcinogenic. Upon metabolic activation, arylhydrazines are transformed into aryl radicals, forming 8-arylpurines, which may play a role in arylhydrazine carcinogenesis. These adducts are poorly read and inhibit chain extension but do alter the conformational preferences of oligonucleotides. We have shown that C8-phenylguanine modification of d(CGCGCG*CGCG) (G*= 8-phenylguanine) stabilizes it in the Z-DNA conformation (B/Z-DNA=1:1, 200 mM NaCl, pH 7.4). Here we have conducted molecular dynamics and free energy calculations to determine the sources(s) of these conformational affects and to predict the affect of the related C8- tolyl and C8-hydroxymethylphenyl guanine adducts on B/Z-DNA equilibrium. Force field parameters for the modified guanines were first developed using Guassian98 employing the B3LYP method and the standard 6–31G* basis set and fit to the Cornell 94 force field with RESP. Molecular dynamics simulations and free energy calculations, using the suite of programs contained in Amber 6 and 7 with the Cornell 94 force field, were used to determine the structural and thermodynamic properties of the DNA. The principal factors that drive conformation are stacking of the aryl group over the 5′-cytosine in the phenyl and tolyl modified oligonucleotides while hydrogen bonding opposes stacking in the hydroxymethylphenyl derivative. The phenyl and tolyl-modified DNA's favored the Z-DNA form as did the hydroxymethylphenyl derivative when hydrogen bonding was not present. The B-DNA conformation was preferred by the unmodified oligonucleotide and by the hydroxymethylphenyl-modified oligonucleotide when hydrogen bonding was considered. Z-DNA stability was not found to directly correlated with carcinogenicity and additional biological factors, such as recognition and repair, may also need to be considered in addition to Z-DNA formation.     

Molecular modeling of the three-dimensional structure of GLP-1R and its interactions with several agonists

Glucagon-like peptide-1 receptor (GLP-1R) is a promising molecular target for developing drugs treating type 2 diabetes. We have predicted the complete three-dimensional structure of GLP-1R and the binding modes of several GLP-1R agonists, including GLP-1, Boc5, and Cpd1, through a combination of homology modeling, molecular docking, and long-time molecular dynamics simulation on a lipid bilayer. Our model can reasonably interpret the results of a number of mutation experiments regarding GLP-1R as well as the successful modification to GLP-1 by Liraglutide. Our model is also validated by a recently revealed crystal structure of the extracellular domain of GLP-1R. An activation mechanism of GLP-1R agonists is proposed based on the principal component analysis and normal mode analysis on our predicted GLP-1R structure. Before the complete structure of GLP-1R is determined through experimental means, our model may serve as a valuable reference for characterizing the interactions between GLP-1R and its agonists. Figure Comparison of our predicted model of rGLP-1R (left) with the recently revealed crystal structure of hGLP-1R (right)     

A Molecular Dynamics Study of the Repressor/Operator(OR1,OR3) Complexes from Bacteriophage 434

We have performed three molecular dynamics simulations using the CHARMM molecular modeling program to study the repressor protein from bacteriophage 434 complexed with DNA operators of two different sequences. Two approaches to the modeling of the solvent were used. In the first method, applied to the R1-69/OR1 truncated complex, water molecules were included explicitly in conjunction with a stochastic boundary force to solvate the complex. In the second approach, used for simulations of the R1-69/OR1 and the R1-69/OR3 complexes, the solvent was omitted and implicitly represented by using a distance-dependent dielectric constant and a scaling of the charges on the exposed residues. The simulation with the model which explicitly includes the solvent serves as a validation of the simulations using a simpler solvent representation. In our discussion of the results we focus upon the important interactions between the DNA binding motif of the 434 repressor (motif helix turn helix) and the operators and how the structures of the complexes change with time.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089460020427     

On the Approximation of Solvent Effects on the Conformation and Dynamics of Cyclosporin A by Stochastic Dynamics Simulation Techniques

Abstract

The molecular simulation technique of stochastic dynamics (SD) is tested by application to the immunosuppressive drug cyclosporin A (CPA). Two stochastic dynamics simulations are performed, one (SD_CCl4 ) with atomic friction coefficients proportional to the viscosity of the nonpolar solvent CCl₄, and one (SD_H2O) with atomic friction coefficients corresponding to an aqueous solution. The atomic friction coefficients are also taken proportional to an approximate expression for the atomic accessible surface area. The properties of both stochastic dynamics simulations are compared to those of two full molecular dynamics (MD) simulations of cyclosporin A, one in a box with 591 CCl₄ molecules, and one in a box with 632 H₂O molecules.

The properties of cyclosporin A as found in the molecular dynamics simulation in CCl₄ are well reproduced by the SD_CCl4 simulation. This indicates that the neglect of a mean force reresenting the average solvent effects on the solute is justified in the case of nonpolar solvents. For polar solvents, like water, this mean force may not be neglected. The SD_H2O simulation of cyclosporin A clearly fails to reproduce the amount of hydrogen bonding found in the molecular dynamics stimulation of cyclosporin A in water.

A comparison with a molecular dynamics simulation of cyclosporin A in vacuo shows that both the SD_CCl4 and the SD_H2O simulation come closer to the properties of the molecular dynamics simulations in CCl₄ and in H₂O than a molecular dynamics simulation in vacuo.     

From Molecular to Process Simulation: Novel Approaches to the Prediction of Phase Equilibria and PVT Behavior Based on Molecular/Quantum Mechanics and Molecular Dynamics Simulations

Abstract

Actually, in modern process simulators, more than 75% of the code implemented is dedicated to physical properties estimation, calculation and predictions. Data banks storing pure component parameters and binary interaction parameters for phase equilibrium calculations are extensively used and continuously implemented in actual process simulators. This gives an idea of the important role physical properties availability plays in process simulation.

In this paper we propose a new way for coupling molecular and process simulation. The basic machinery is to resort to molecular/quantum mechanics and molecular dynamics simulation techniques for generating the parameters of some equations of state that will subsequently be used for the prediction of phase equilibria and PVT behavior of small and polymeric molecules as well. This information, in turn, will be used as input in the process simulator, thus creating a final and well-defined bridge between molecular and process simulations in chemical engineering.     

Molecular Dynamics Simulation in Solvent of the Estrogen Receptor Protein DNA Binding Domain in Complex with a Non-Consensus Estrogen Response Element DNA Sequence

Abstract

We investigated protein/DNA interactions, using molecular dynamics simulations computed between a 10 Angstom water layer model of the estrogen receptor (ER) protein DNA binding domain (DBD) amino acids and DNA of a non-consensus estrogen response element (ERE) consisting of 29 nucleotide base pairs. This ERE nucleotide sequence occurs naturally upstream of the Xenopus laevis Vitelligenin AI gene. The ER DBD is encoded by three exons. Namely, exons 2 and 3 which encode the two zinc binding motifs and a sequence of exon 4 which encodes a predicted alpha helix. We generated a computer model of the ER DBD using atomic coordinates derived from the average of 30 nuclear magnetic resonance (NMR) spectroscopy coordinate sets. Amino acids on the carboxyl end of the ER DBD were disordered in both X-ray crystallography and NMR determinations and no coordinates were reported. This disordered region includes 10 amino acids of a predicted alpha helix encoded in exon 4 at the exon 3/4 splice junction. These amino acids are known to be important in DNA binding and are also believed to function as a nuclear translocation signal sequence for the ER protein. We generated a computer model of the predicted alpha helix consisting of the 10 amino acids encoded in exon 4 and attached this helix to the carboxyl end of the ER DBD at the exon 3/4 splice junction site. We docked the ER DBD model within the DNA major groove halfsites of the 29 base pair non-consensus ERE and flanking nucleotides. We constructed a solvated model with the ER DBD/ERE complex surrounded by a ten Angstrom water layer and conducted molecular dynamics simulations. Hydrogen bonding interactions were monitored. In addition, van der Waals and electrostatic interaction energies were calculated. Amino acids of the ER DBD DNA recognition helix formed both direct and water mediated hydrogen bonds at cognate codon-anticodon nucleotide base and backbone sites within the ERE DNA right major groove halfsite. Amino acids of the ER DBD exon 4 encoded predicted alpha helix formed direct and water mediated H-bonds with base and backbone sites of their cognate codon-anticodon nucleotides within the minor grooves flanking the ERE DNA major groove halfsites. These interactions together induced bending of the DNA into the protein.     

Disruption Mechanism in the Helix of SPF Peptide by Interchanging E5 and K10 Residues: Inference from Molecular Dynamics Study

Abstract

Seminalplasmin (SPLN) is a 47-residue peptide (SDEKASPDKHHRFSLSRYAKLANR LANPKLLETFLSKWIGDRGNRSV) from bovine seminal plasma. It has broad spectrum antimicrobial activity, without any hemolytic activity. The 28–40 segment of SPLN with the sequence PKLLETFLSKWIG, designated as SPF, is the most hydrophobic stretch of SPLN and primarily responsible for the membrane-perturbing activity of SPLN. It was reported that SPF has a helical structure and the interchange of E5 and K10 residues disrupted the helical structure. The present paper reports a possible mechanism of disruption of the helical structure of SPF peptide during the interchange of E5 and K10 residues. The result is based on simulated annealing and molecular dynamics simulation studies on SPF and its four analogues with K10E, K10D, E5K, and E5K & K10E substitutions. It showed that K10 residue has a critical role in maintaining the highest helical content and the positions of charged residues are also very important for maintaining the helical structure of the SPF peptide. Formation of some new long-range hydrogen bonds and the rupture of some shortrange hydrogen bonds involving the tenth residue led to the disruption of helical structure of SPF peptide when E5 and K10 residues are interchanged.     

The Implementation of the Iterative Diagonalization Scheme and ab initio Molecular Dynamics Simulation with the LAPW Method

Abstract

A new ab initio molecular dynamics method based on the full-potential linearized-augmented-plane-wave (LAPW) basis set has been implemented. The LAPW basis set has been successfully employed for systems containing localized electrons such as first row atoms and transition metals. In our implementation of the LAPW-MD scheme, iterative residual minimization algorithm is used to solve the electronic states problem. The atoms are moved according to forces derived from the Hellman–Feynman theorem and incomplete basis set correction terms. The performance of the program is further enhanced by parallelization. We will discuss technical details of the program implementation and present results obtained from this code to the equilibrium structures and vibrational properties of simple diatomic molecules.     

Molecular Dynamics Simulations on Pars Intercerebralis Major Peptide-C (PMP-C) Reveal the Role of Glycosylation and Disulfide Bonds in its Enhanced Structural Stability and Function

Abstract

Fucosylation of Thr 9 in pars intercerebralis major peptide-C (PMP-C) enhances its structural stability and functional ability as a serine protease inhibitor. In order to understand the role of disulfide bonds and glycosylation on the structure and function of PMP-C, we have carried out multiple explicit solvent molecular dynamics (MD) simulations on fucosylated and non-fucosylated forms of PMP-C, both in the presence and absence of the disulfide bonds. Our simulations revealed that there were no significant structural changes in the native disulfide bonded forms of PMP-C due to fucosylation. On the other hand, the non-fucosylated form of PMP-C without disulfide bonds showed larger deviations from the starting structure than the fucosylated form. However, the structural deviations were restricted to the terminal regions while core β-sheet retained its hydrogen bonded structure even in absence of disulfide bonds as well as fucosylation. Interestingly, fucosylation of disulfide bonded native PMP-C led to a decreased thermal flexibility in the residue stretch 29–32 which is known to interact with the active site of the target proteases. Our analysis revealed that disulfide bonds covalently connect the residue stretch 29–32 to the central β-sheet of PMP-C and using a novel network of side chain interactions and disulfide bonds fucosylation at Thr 9 is altering the flexibility of the stretch 29–32 located at a distal site. Thus, our simulations explain for the first time, how presence of disulfide bonds between conserved cysteines and fucosylation enhance the function of PMP-C as a protease inhibitor.     

Molecular dynamics studies on HIV-1 protease: a comparison of the flap motions between wild type protease and the M46I/G51D double mutant

The emergence of drug-resistant mutants of HIV-1 is a tragic effect associated with conventional long-treatment therapies against acquired immunodeficiency syndrome. These mutations frequently involve the aspartic protease encoded by the virus; knowledge of the molecular mechanisms underlying the conformational changes of HIV-1 protease mutants may be useful in developing more effective and longer lasting treatment regimes. The flap regions of the protease are the target of a particular type of mutations occurring far from the active site. These mutations modify the affinity for both substrate and ligands, thus conferring resistance. In this work, molecular dynamics simulations were performed on a native wild type HIV-1 protease and on the drug-resistant M46I/G51D double mutant. The simulation was carried out for a time of 3.5 ns using the GROMOS96 force field, with implementation of the SPC216 explicit solvation model. The results show that the flaps may exist in an ensemble of conformations between a “closed” and an “open” conformation. The behaviour of the flap tips during simulations is different between the native enzyme and the mutant. The mutation pattern leads to stabilization of the flaps in a semi-open configuration.     

A QM/MM study of the complexes formed by aluminum and iron with serum transferrin at neutral and acidic pH

Serum transferrin (sTf) transports iron in serum and internalizes in cells via receptor mediated endocytosis. Additionally, sTf has been identified as the predominant aluminum carrier in serum. Some questions remain unclear about the exact mechanism for the metal release or whether the aluminum and iron show the same binding mode during the entire process. In the present work, simulation techniques at quantum and atomic levels have been employed in order to gain access into a molecular level understanding of the metal-bound sTf complex, and to describe the binding of Al(III) and Fe(III) ions to sTf. First, hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations were carried out in order to analyze the dynamics of the aluminum-loaded complex, taking into account the different pH conditions in blood and into the cell. Moreover, the complexes formed by transferrin with Al(III) and Fe(III) were optimized with high level density functional theory (DFT)/MM methods. All these results indicate that the interaction mode of Al(III) and Fe(III) with sTf change upon different pH conditions, and that the coordination of Al(III) and Fe(III) is not equivalent during the metal intake, transport and release processes. Our results emphasize the importance of the pH on the metal binding and release mechanism and suggest that Al(III) can follow the iron pathway to get access into cells, although once there, it may show a different binding mode, leading to a different mechanism for its release.     

Insights Into the Enzymatic Mechanism of 6-Phosphogluconolactonase from Trypanosoma brucei Using Structural Data and Molecular Dynamics Simulation

Trypanosoma brucei is the causative agent of African sleeping sickness. Current work for the development of new drugs against this pathology includes evaluation of enzymes of the pentose phosphate pathway (PPP), which first requires a clear understanding of their function and mechanism of action. In this context, we focused on T. brucei 6-phosphogluconolactonase (Tb6PGL), which converts δ-6-phosphogluconolactone into 6-phosphogluconic acid in the second step of the PPP. We have determined the crystal structure of Tb6PGL in complex with two ligands, 6-phosphogluconic acid and citrate, at 2.2 Å and 2.0 Å resolution, respectively. We have performed molecular dynamics (MD) simulations on Tb6PGL in its empty form and in complex with δ-6-phosphogluconolactone, its natural ligand. Analysis of the structural data and MD simulations allowed us to propose a detailed enzymatic mechanism for 6PGL enzymes.     

Inhibition of Oncogenic and Activated Wild-Type ras-p21 Protein-Induced Oocyte Maturation by Peptides from the ras-Binding Domain of the raf-p74 Protein, Identified from Molecular Dynamics Calculations

In the preceding paper we found from molecular dynamics calculations that the structure of the ras-binding domain (RBD) of raf changes predominantly in three regions depending upon whether it binds to ras-p21 protein or to its inhibitor protein, rap-1A. These three regions of the RBD involve residues from the protein–protein interaction interface, e.g., between residues 60 and 72, residues 97–110, and 111–121. Since the rap-1A–RBD complex is inactive, these three regions are implicated in ras-p21-induced activation of raf. We have therefore co-microinjected peptides corresponding to these three regions, 62–76, 97–110, and 111–121, into oocytes with oncogenic p21 and microinjected them into oocytes incubated in in insulin, which activates normal p2l. All three peptides, but not a control peptide, strongly inhibit both oncogenic p21- and insulin-induced oocyte maturation. These findings corroborate our conclusions from the theoretical results that these three regions constitute raf effector domains. Since the 97–110 peptide is the strongest inhibitor of oncogenic p21, while the 111–121 peptide is the strongest inhibitor of insulin-induced oocyte maturation, the possibility exists that oncogenic and activated normal p21 proteins interact differently with the RBD of raf.     

Molecular Dynamics on Complexes of ras-p21 and Its Inhibitor Protein, rap-1A, Bound to the ras-Binding Domain of the raf-p74 Protein: Identification of Effector Domains in the raf Protein

We have computed the average structures for the ras-p21 protein and its strongly homologous inhibitor protein, rap-1A, bound to the ras-binding domain (RBD) of the raf protein, using molecular dynamics. Our purpose is to determine the differences in structure between these complexes that would result in no mitogenic activity of rap-1A-RBD but full activity of p21-RBD. We find that despite the similarities of the starting structures for both complexes, the average structures differ considerably, indicating that these two proteins do not interact in the same way with this vital target protein. p21 does not undergo major changes in conformation when bound to the RBD, while rap-1 A undergoes significant changes in structure on binding to the RBD, especially in the critical region around residue 61. The p21 and rap-1A make substantially different contacts with the RBD. For example, the loop region from residues 55–71 of rap-la makes extensive hydrogen-bond contacts with the RBD, while the same residues of p21 do not. Comparison of the structures of the RBD in both complexes reveals that it undergoes considerable changes in structure when its structure bound to p21 is compared with that bound to rap-1A. These changes in structure are due to displacements of regular structure (e.g., -helices and -sheets) rather than to changes in the specific conformations of the segments themselves. Three regions of the RBD have been found to differ significantly from one another in the two complexes: the binding interface between the two proteins at residues 60 and 70, the region around residues 105–106, and 118–120. These regions may constitute effector domains of the RBD whose conformations determine whether or not mitogenic signal transduction will occur.     

Molecular analysis of the split cox1 gene from the Basidiomycota Agrocybe aegerita: relationship of its introns with homologous Ascomycota introns and divergence levels from common ancestral copies

The Basidiomycota Agrocybe aegerita (Aa) mitochondrial cox1 gene (6790 nucleotides), encoding a protein of 527 aa (58 377 Da), is split by four large subgroup IB introns possessing site-specific endonucleases assumed to be involved in intron mobility. When compared to other fungal COX1 proteins, the Aa protein is closely related to the COX1 one of the Basidiomycota Schizophyllum commune (Sc). This clade reveals a relationship with the studied Ascomycota ones, with the exception of Schizosaccharomyces pombe (Sp) which ranges in an out-group position compared with both higher fungi divisions. When comparison is extended to other kingdoms, fungal COX1 sequences are found to be more related to algae and plant ones (more than 57.5% aa similarity) than to animal sequences (53.6% aa similarity), contrasting with the previously established close relationship between fungi and animals, based on comparisons of nuclear genes. The four Aa cox1 introns are homologous to Ascomycota or algae cox1 introns sharing the same location within the exonic sequences. The percentages of identity of the intronic nucleotide sequences suggest a possible acquisition by lateral transfers of ancestral copies or of their derived sequences. These identities extend over the whole intronic sequences, arguing in favor of a transfer of the complete intron rather than a transfer limited to the encoded ORF. The intron i4 shares 74% of identity, at the nucleotidic level, with the Podospora anserina (Pa) intron i14, and up to 90.5% of aa similarity between the encoded proteins, i.e. the highest values reported to date between introns of two phylogenetically distant species. This low divergence argues for a recent lateral transfer between the two species. On the contrary, the low sequence identities (below 36%) observed between Aa i1 and the homologous Sp i1 or Prototheca wickeramii (Pw) i1 suggest a long evolution time after the separation of these sequences. The introns i2 and i3 possessed intermediate percentages of identity with their homologous Ascomycota introns. This is the first report of the complete nucleotide sequence and molecular organization of a mitochondrial cox1 gene of any member of the Basidiomycota division.     

Mechanisms for L-channel-mediated increase in [Ca]i and its reduction by anti-bipolar drugs in cultured astrocytes combined with its mRNA expression in freshly isolated cells support the importance of astrocytic L-channels

The importance of Ca²⁺ signaling in astrocytes is undisputed but a potential role of Ca²⁺ influx via L-channels in the brain in vivo is disputed, although expression of these channels in cultured astrocytes is recognized. This study shows that an increase in free cytosolic Ca²⁺ concentration ([Ca²⁺]_i) in astrocytes in primary cultures in response to an increased extracellular K⁺ concentration (45 mM) is inhibited not only by nifedipine (confirming previous observations) but also to a very large extent by ryanodine, inhibiting ryanodine receptor-mediated release of Ca²⁺, known to occur in response to an elevation in [Ca²⁺]_i. This means that the actual influx of Ca²⁺ is modest, which may contribute to the difficulty in demonstrating L-channel-mediated Ca²⁺ currents in astrocytes in intact brain tissue. Chronic treatment with any of the 3 conventional anti-bipolar drugs lithium, carbamazepine or valproic acid similarly causes a pronounced inhibition of K⁺-mediated increase in [Ca²⁺]_i. This is shown to be due to an inhibition of capacitative Ca²⁺ influx, reflected by decreased mRNA and protein expression of the ‘transient receptor potential channel’ (TRPC1), a constituent of store-operated channels (SOCEs). Literature data are cited (i) showing that depolarization-mediated Ca²⁺ influx in response to an elevated extracellular K⁺ concentration is important for generation of Ca²⁺ oscillations and for the stimulatory effect of elevated K⁺ concentrations in intact, non-cultured brain tissue, and (ii) that Ca²⁺ channel activity is dependent upon availability of metabolic substrates, including glycogen. Finally, expression of mRNA for Ca_v1.3 is demonstrated in freshly separated astrocytes from normal brain.     

Structure of cDNA clones for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase from a fern (Asplenium cataractarum rosenstock,aspleniaceae), and its comparison with those of various seed plants

We isolated the small subunit of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO SSu) from a fern,Asplenium cataractarum and determined its 34 N-terminal amino acid sequence. We obtained a cDNA clone that contains the entire coding region of the SSu from the same fern species, using synthetic oligonucleotide probes derived from the above amino acid sequence. It contains a 525 bp open reading frame capable of coding for a polypeptide with 174 amino acids, 31 bp 5′-and 206 bp 3′-noncoding regions. It was also elucidated that the precursor to the SSu contains a transit peptide of 53 amino acid residues and a mature protein of 121 residues. We compared the deduced amino acid sequence of the fern SSu with those of 11 other vascular plant species (including gymnosperms, monocots and dicots). As low as 55% homology was observed between those of a fern and seed plants. Constancy of the amino acid substitution rate in RuBisCO SSu was supported by our relative rate test. Amino acid substitution rate per year per site for RuBisCO SSu was calculated to be 0.81×10⁻⁹ assuming that the separation between pteridophytes and seed plants arose 380 million years ago.     

Interaction of N,N,N′,N′-tetramethyl-p-phenylenediamine with photosystem II as revealed by thermoluminescence: Reduction of the higher oxidation states of the Mn cluster and displacement of plastoquinone from the QB niche

The flash-induced thermoluminescence (TL) technique was used to investigate the action of N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) on charge recombination in photosystem II (PSII). Addition of low concentrations (μM range) of TMPD to thylakoid samples strongly decreased the yield of TL emanating from S₂Q_B⁻ and S₃Q_B⁻ (B-band), S₂Q_A⁻ (Q-band), and Y_D⁺Q_A⁻ (C-band) charge pairs. Further, the temperature-dependent decline in the amplitude of chlorophyll fluorescence after a flash of white light was strongly retarded by TMPD when measured in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Though the period-four oscillation of the B-band emission was conserved in samples treated with TMPD, the flash-dependent yields (Y_n) were strongly declined. This coincided with an upshift in the maximum yield of the B-band in the period-four oscillation to the next flash. The above characteristics were similar to the action of the ADRY agent, carbonylcyanide m-chlorophenylhydrazone (CCCP). Simulation of the B-band oscillation pattern using the integrated Joliot-Kok model of the S-state transitions and binary oscillations of Q_B confirmed that TMPD decreased the initial population of PSII centers with an oxidized plastoquinone molecule in the Q_B niche. It was deduced that the action of TMPD was similar to CCCP, TMPD being able to compete with plastoquinone for binding at the Q_B-site and to reduce the higher S-states of the Mn cluster.