Insights into the protonation state and spin structure for the g = 2 multiline electron paramagnetic resonance signal of the oxygen-evolving complex.

In photosystem II (PSII), one-electron oxidation of the most stable oxidation state of the Mn4CaO5 cluster (S1) leads to formation of two distinct states, the open-cubane S2 conformation [Mn1(III)Mn2(IV)Mn3(IV)Mn4(IV)] with low spin and the closed-cubane S2 conformation [Mn1(IV)Mn2(IV)Mn3(IV)Mn4(III)] with high spin. In electron paramagnetic resonance (EPR) spectroscopy, the open-cubane S2 conformation exhibits a g = 2 multiline signal. However, its protonation state remains unclear. Here, we investigated the protonation state of the open-cubane S2 conformation by calculating exchange couplings in the presence of the PSII protein environment and simulating the pulsed electron-electron double resonance (PELDOR). When a ligand water molecule, which forms an H-bond with D1-Asp61 (W1), is deprotonated at dangling Mn4(IV), the first-exited energy (34 cm-1) in manifold spin excited states aligns with the observed value in temperature-dependent pulsed EPR analyses, and the PELDOR signal is best reproduced. Consequently, the g = 2 multiline signal observed in EPR corresponds to the open-cubane S2 conformation with the deprotonated W1 (OH-).

Molecular Structure Related to an S = 5/2 High-Spin S2 State Manganese Cluster of Photosystem II Investigated by Q-Band Pulse EPR Spectroscopy.

The high-spin S2 state of the photosynthetic oxygen-evolving cluster Mn4CaO5, corresponding to the g = 4.1 signal for X-band electron paramagnetic resonance (EPR), was investigated using Q-band pulsed EPR, which detected a main peak at g = 3.10 and satellite peaks at 5.25, 4.55, and 2.80. We evaluated the spin state as the zero-field splitting of D = 0.465 cm-1 and E/D = 0.245 with S = 5/2. The temperature dependence of the T1 relaxation time revealed that the excited-state energy was 28.7 cm-1 higher than that of the S = 5/2 ground state. By comparing present quantum mechanical (QM) calculation models, a closed-cubane structure with the protonation state of two oxygens, W1 (= OH-) and W2 (= H2O), was the most probable structure for the S = 5/2 state. The three-pulse electron spin-echo envelope modulation (ESEEM) detected the nuclear signal, which was assigned to nitrogen as His332 ligated to the Mn1 ion. The obtained hyperfine constant for the nitrogen signal was significantly reduced from that in the S = 1/2 low-spin state. These results indicate that the S = 5/2 spin state arises from the closed-cubane structure.

A [4Fe-4S] cluster resides at the active center of phosphomevalonate dehydratase, a key enzyme in the archaeal modified mevalonate pathway.

The recent discovery of the archaeal modified mevalonate pathway revealed that the fundamental units for isoprenoid biosynthesis (isopentenyl diphosphate and dimethylallyl diphosphate) are biosynthesized via a specific intermediate, trans-anhydromevalonate phosphate. In this biosynthetic pathway, which is unique to archaea, the formation of trans-anhydromevalonate phosphate from (R)-mevalonate 5-phosphate is catalyzed by a key enzyme, phosphomevalonate dehydratase. This archaea-specific enzyme belongs to the aconitase X family within the aconitase superfamily, along with bacterial homologs involved in hydroxyproline metabolism. Although an iron-sulfur cluster is thought to exist in phosphomevalonate dehydratase and is believed to be responsible for the catalytic mechanism of the enzyme, the structure and role of this cluster have not been well characterized. Here, we reconstructed the iron-sulfur cluster of phosphomevalonate dehydratase from the hyperthermophilic archaeon Aeropyrum pernix to perform biochemical characterization and kinetic analysis of the enzyme. Electron paramagnetic resonance, iron quantification, and mutagenic studies of the enzyme demonstrated that three conserved cysteine residues coordinate a [4Fe-4S] cluster-as is typical in aconitase superfamily hydratases/dehydratases, in contrast to bacterial aconitase X-family enzymes, which have been reported to harbor a [2Fe-2S] cluster.

Extremely low-frequency pulses of faint magnetic field induce mitophagy to rejuvenate mitochondria.

Humans are frequently exposed to time-varying and static weak magnetic fields (WMF). However, the effects of faint magnetic fields, weaker than the geomagnetic field, have been scarcely reported. Here we show that extremely low-frequency (ELF)-WMF, comprised of serial pulses of 10 µT intensity at 1-8 Hz, which is three or more times weaker than the geomagnetic field, reduces mitochondrial mass to 70% and the mitochondrial electron transport chain (ETC) complex II activity to 88%. Chemical inhibition of electron flux through the mitochondrial ETC complex II nullifies the effect of ELF-WMF. Suppression of ETC complex II subsequently induces mitophagy by translocating parkin and PINK1 to the mitochondria and by recruiting LC3-II. Thereafter, mitophagy induces PGC-1α-mediated mitochondrial biogenesis to rejuvenate mitochondria. The lack of PINK1 negates the effect of ELF-WMF. Thus, ELF-WMF may be applicable for the treatment of human diseases that exhibit compromised mitochondrial homeostasis, such as Parkinson's disease.

Temperature dependence of the formation of the g ~ 5 EPR signal in the oxygen evolving complex of photosystem II.

The temperature dependence of the formation of the g ~ 5 S2 state electron paramagnetic resonance (EPR) signal in photosystem II (PSII) was investigated. The g ~ 5 signal was produced at an illumination above 200 K. The half inhibition temperature of the formation of the g ~ 5 EPR signal was approximately 215 K. The half inhibition temperature is close to that of the transition from the S2 state-to-S3 state in the untreated PSII, and not to that of the transition from S1 state -to-S2 state in the untreated PSII. The upshift of the half inhibition temperature of the transition from the S1 state -to-S2 state (g ~ 5) reflects the structural change upon transition from the S1 state to the S2 state. The activation energy of the g ~ 5 state formation was estimated as 40.7 ± 4.4 kJ/mol, which is comparable to the reported activation energy for the S2 formation in the untreated PSII. The activation enthalpy and entropy were estimated to be 39.0 ± 4.4 kJ/mol and - 103 ± 19 J/mol K at 210 K, respectively. Based on these parameters, the formation process of the g ~ 5 state is discussed in this study.

Pulse-frequency-dependent resonance in a population of pyramidal neuron models.

Stochastic resonance is known as a phenomenon whereby information transmission of weak signal or subthreshold stimuli can be enhanced by additive random noise with a suitable intensity. Another phenomenon induced by applying deterministic pulsatile electric stimuli with a pulse frequency, commonly used for deep brain stimulation (DBS), was also shown to improve signal-to-noise ratio in neuron models. The objective of this study was to test the hypothesis that pulsatile high-frequency stimulation could improve the detection of both sub- and suprathreshold synaptic stimuli by tuning the frequency of the stimulation in a population of pyramidal neuron models. Computer simulations showed that mutual information estimated from a population of neural spike trains displayed a typical resonance curve with a peak value of the pulse frequency at 80-120 Hz, similar to those utilized for DBS in clinical situations. It is concluded that a "pulse-frequency-dependent resonance" (PFDR) can enhance information transmission over a broad range of synaptically connected networks. Since the resonance frequency matches that used clinically, PFDR could contribute to the mechanism of the therapeutic effect of DBS.

Location of two Mn2+ affinity sites in photosystem II detected by pulsed electron-electron double resonance.

In this study, we identified two Mn2+ sites in apo-Photosystem II (PSII) using the pulsed electron-electron double resonance (PELDOR). A Mn2+ ion was bound to apo-PSII on the deactivation of the oxygen-evolving complex. The electron-electron magnetic dipole interaction of the Mn2+ to YD· was estimated to be 2.4 MHz. The site was assigned at the position between His332 and Glu189 in the D1 polypeptide, which is close to the Mn1 site in mature PS II. Using recent structures observed under electron microscopes (EM), the location of the Mn2+ site on photoactivation was reevaluated. The position between Asp170 and Glu189 in the D1 polypeptide is a good candidate for the initial high-affinity site for photoactivation. Based on a comparison with the PELDOR results, the two EM structures were evaluated.

Protonation structure of the closed-cubane conformation of the O2-evolving complex in photosystem II.

In photosystem II (PSII), one-electron oxidation of the most stable state of the oxygen-evolving Mn4CaO5 cluster (S1) leads to the S2 state formation, Mn1(III)Mn2(IV)Mn3(IV)Mn4(IV) (open-cubane S2) or Mn1(IV)Mn2(IV)Mn3(IV)Mn4(III) (closed-cubane S2). In electron paramagnetic resonance (EPR) spectroscopy, the g = 4.1 signal is not observed in cyanobacterial PSII but in plant PSII, whereas the g = 4.8 signal is observed in cyanobacterial PSII and extrinsic-subunit-depleted plant PSII. Here, we investigated the closed-cubane S2 conformation, a candidate for a higher spin configuration that accounts for g > 4.1 EPR signal, considering all pairwise exchange couplings in the PSII protein environment (i.e. instead of considering only a single exchange coupling between the [Mn3(CaO4)] cubane region and the dangling Mn4 site). Only when a ligand water molecule that forms an H-bond with D1-Asp61 (W1) is deprotonated at dangling Mn4(IV), the g = 4.1 EPR spectra can be reproduced using the cyanobacterial PSII crystal structure. The closed-cubane S2 is less stable than the open-cubane S2 in cyanobacterial PSII, which may explain why the g = 4.1 EPR signal is absent in cyanobacterial PSII.

The evolutionary conserved iron-sulfur protein TCR controls P700 oxidation in photosystem I.

In natural habitats, plants have developed sophisticated regulatory mechanisms to optimize the photosynthetic electron transfer rate at the maximum efficiency and cope with the changing environments. Maintaining proper P700 oxidation at photosystem I (PSI) is the common denominator for most regulatory processes of photosynthetic electron transfers. However, the molecular complexes and cofactors involved in these processes and their function(s) have not been fully clarified. Here, we identified a redox-active chloroplast protein, the triplet-cysteine repeat protein (TCR). TCR shared similar expression profiles with known photosynthetic regulators and contained two triplet-cysteine motifs (CxxxCxxxC). Biochemical analysis indicated that TCR localizes in chloroplasts and has a [3Fe-4S] cluster. Loss of TCR limited the electron sink downstream of PSI during dark-to-light transition. Arabidopsis pgr5-tcr double mutant reduced growth significantly and showed unusual oxidation and reduction of plastoquinone pool. These results indicated that TCR is involved in electron flow(s) downstream of PSI, contributing to P700 oxidation.

Formation of the High-Spin S2 State Related to the Extrinsic Proteins in the Oxygen Evolving Complex of Photosystem II.

The high-spin S2 state was investigated with photosystem II (PSII) from spinach, Thermosynechococcus vulcanus, and Cyanidioschyzon merolae. In extrinsic protein-depleted PSII, high-spin electron paramagnetic resonance (EPR) signals were not detected in either species, whereas all species showed g ∼ 5 signals in the presence of a high concentration of Ca2+ instead of the multiline signal. In the intact and PsbP/Q-depleted PSII from spinach, the g = 4.1 EPR signal was detected. These results show that formation of the high-spin S2 state of the manganese cluster is regulated by the extrinsic proteins through a charge located near the Mn4 atom in the Mn4CaO5 cluster but is independent of the intrinsic proteins. The shift to the g ∼ 5 state is caused by tilting of the z-axis in the Mn4 coordinates through hydrogen bonds or external divalent cations. The structural modification may allow insertion of an oxygen atom during the S2-to-S3 transition.

Molecular Structure of the S2 State with a g = 5 Signal in the Oxygen Evolving Complex of Photosystem II.

The g-factor shift of the g = 4.1 EPR signal was detected in spinach PsbO/P/Q-depleted PS II. The effective g-factor of the signal shifts up to ∼4.9, depending on the Ca2+ concentration. Hyperfine structure spacing with about 3 mT was detected in this g = 5 (4.9) signal. The shift to g = 5 (4.9) was related to the distortion of the manganese cluster, derived from the modification of the chemical bond or the crystalline field of the Mn4(III) in the manganese cluster. Based on the EPR analysis of the g = 5 (4.9) spin state, another molecular structure of the S2 state, a "distant Mn" structure, was discussed as an intermediate state between the S2 and S3 states.

Orientation of Ligand Field for Dangling Manganese in Photosynthetic Oxygen-Evolving Complex of Photosystem II.

The spin structure in the S2 state and the crystal structure of the manganese cluster of the oxygen-evolving complex of plant photosystem II were combined by the quantitative evaluation of the magnetic anisotropy of the g = 4 signal. The g-values of 3.93 and 4.13 were obtained for the g = 4 signal in the directions parallel and perpendicular to the membrane normal, respectively. The peak-to-peak separations were 270 and 420 G for the parallel and perpendicular orientations to the membrane, respectively. By comparison with the crystal structure, the z-axis of the zero-field splitting was ascribed to the direction of the dangling Mn connecting water oxygen, Mn4-O(W1), in the manganese cluster. The results give the first experimental evidence that the valence of the dangling Mn is Mn(III) in the S2 high-spin state. We showed that the strong exchange coupling of Mn4 to Mn3 was required for g = 4.1 spin state in the four-spin couplings, estimated as > ∼|-30 cm-1|, indicating that the present closed cubane model in quantum mechanics/molecular mechanics (QM/MM) calculation cannot explain the g = 4.1 spin structure. The onsite zero-field splitting of the dangling Mn was evaluated as -2.3 cm-1 under the strong antiferromagnetic couplings (-50 cm-1) with the dangling Mn to the cubane frame in the four coupled spin state. From the viewpoint of the arrangement of the Mn valences in the cluster, a closed cubane model is effective, but without a large structural deviation from the S1 state crystal structure.

Modeling of spike trains in auditory nerves with self-exciting point processes of the von Mises type.

This article presents the modeling of spike trains in auditory nerve fiber (ANF) models with a one-memory self-exciting point process (SEPP) of the von Mises type. The ANF models were acoustically stimulated by a synaptic current of inner hair cells, or electrically stimulated by sinusoidally amplitude-modulated pulsatile waveforms. It has been shown that the parameters of one-memory SEPP of the von Mises type could be estimated by numerically maximizing the likelihood function from sample realizations of the spike trains in response to acoustic or electric stimulus. Furthermore, it was found that period histograms of the one-memory SEPP generated artificially on the basis of the estimated von Mises parameters agreed well with those of acoustic or electric stimulus, by performing the uniform-scores test. It implies that the waveforms of pulsatile electric stimuli should be selected such that the spike trains can be represented by one-memory SEPP of the von Mises type with appropriate parameters, efficiently carrying information to the cochlear implant user's brain, like that in acoustic stimulation of the healthy ear. The findings presented in this paper may play an important role in determining optimal parameters of pulsatile electric stimuli by using one-memory SEPP of the von Mises type, and further in the design of better cochlear prostheses.

Flavin-Radical Formation in the Light-Oxygen-Voltage-Sensing Domain of the Photozipper Blue-light Sensor Protein.

Formation of the neutral flavin radical in the light-oxygen-voltage-sensing (LOV-sensing) domain of photozipper, based on VfAUREO1, was investigated by electron paramagnetic resonance spectroscopy. The flavin radical was observed in the presence of dithiothreitol by illumination of a LOV-domain mutant (C254S), in which a photoactive cysteine residue in close proximity to flavin was replaced with a serine. The radical did not form under low initial protein-concentration conditions (less than 20 µM). The flavin radicals accumulated with logistic time-dependent kinetics when the protein concentrations were higher than 30 µM. These results indicate that the radical is produced by concerted reactions involving protein interactions and that the radical is formed from the LOV dimer but not the LOV monomer. In contrast, logistic time dependencies were not observed for the sample adapted to the dark following radical formation by illumination, indicating that initialization of the proton pathway is essential for this fast sensing reaction.

Magnetic structure of manganese cluster in photosystem II investigated by electron paramagnetic resonance.

The electronic structure of manganese (Mn) cluster in photosystem II was investigated by electron paramagnetic resonance (EPR) spectroscopy. In order to determine the spin density distribution in magnetically coupled Mn in the S2 state Mn cluster, pulsed electron-electron double resonance (PELDOR) measurement was performed. The local environment of the Mn cluster was investigated by electron-nuclear double resonance (ENDOR). Using spin projections determined by PELDOR, ENDOR signals were assigned to the water molecules ligated to the Mn cluster. The location of a high-affinity Mn2+ site in apo-photosystem II, which is the initial site of photoactivation of the Mn cluster, was determined by PELDOR.

Location of the extrinsic subunit PsbP in photosystem II studied by pulsed electron-electron double resonance.

The binding site of the extrinsic protein PsbP in plant photosystem II was mapped by pulsed electron-electron double resonance, using mutant spinach PsbP (Pro20Cys, Ser82Cys, Ala111Cys, and Ala186Cys) labeled with 4-maleimido-TEMPO (MSL) spin label. The distances between the spin label and the Tyr160 neutral radical (YD) in PsbD, the D2 subunit of plant photosystem II, were 50.8 ±â€¯3.5 Å, 54.9 ±â€¯4.0 Å, 57.8 ±â€¯4.9 Å, and 58.4 ±â€¯14.1 Å, respectively. The geometry inferred from these distances was fitted to the PsbP crystal structure (PDB: 4RTI) to obtain the coordinates of YD relative to PsbP. These coordinates were then fitted under boundary conditions to the structure of cyanobacterial photosystem II (PDB: 4UB6), by rotating on Euler angles centered at fixed YD coordinates. The result proposed two models which show possible acidic amino acid residues in CP43, CP47 and D2 that can bind the basic amino acids Arg48, Lys143, and Lys160 in PsbP.

Dimeric Structure of the Blue Light Sensor Protein Photozipper in the Active State.

The light oxygen voltage-sensing (LOV) domain plays a crucial role in blue light (BL) sensing in plants and microorganisms. LOV domains are usually associated with the effector domains and regulate the activities of effector domains in a BL-dependent manner. Photozipper (PZ) is monomeric in the dark state. BL induces reversible dimerization of PZ and subsequently increases its affinity for the target DNA sequence. In this study, we report the analyses of PZ by pulsed electron-electron double resonance (PELDOR). The neutral flavin radical was formed by BL illumination in the presence of dithiothreitol in the LOV-C254S (without the bZIP domain) and PZ-C254S mutants, where the cysteine residue responsible for adduct formation was replaced with serine. The magnetic dipole interactions of 3 MHz between the neutral radicals were detected in both LOV-C254S and PZ-C254S, indicating that these mutants are dimeric in the radical state. The PELDOR simulation showed that the distance between the radical pair is close to that estimated from the dimeric crystal structure in the "light state" [Heintz, U., and Schlichting, I. (2016) eLife 5, e11860], suggesting that in the radical state, LOV domains in PZ-C254S form a dimer similar to that of LOV-C254S, which lacks the bZIP domain.

On a unified point process approach for the characterization of bioelectric discrete phenomena.

This paper discusses a unified method based on the theory of point processes to characterize various types of bioelectric discrete signals such as heart beat timing, myoelectric activity, discharge of primary sensory neurons or neurons in the central nervous systems. The doubly stochastic point processes, in which the discrete event occurring intensity is stochastic, forms the most general class to characterize the discrete phenomena. In this paper the self-exciting process has been shown to be useful to characterize wide range of discrete biosignals. The modeling of conditional intensity function is the essential part of the characterization. When the intensity has a parametric model, the maximum likelihood parameter estimation will be the useful way to characterize the phenomena. The effectiveness of the method is demonstrated by a specific modeling of the spontaneous neuronal burst discharges recorded from the brain thalamus during the neuro surgery. The first approximation model has four parameters obtained by the instantaneous nonlinearly transformed sinusoidal function. An extended model allows arbitrary periodic intensity with refractory period. Predicted interval histograms show good agreement with the observed ones indicating the validity of the proposed method.

Mechanism of Photodamage of the Oxygen Evolving Mn Cluster of Photosystem II by Excessive Light Energy.

Photodamage to Photosystem II (PSII) has been attributed either to excessive excitation of photosynthetic pigments or by direct of light absorption by Mn4CaO5 cluster. Here we investigated the time course of PSII photodamage and release of Mn in PSII-enriched membranes under high light illumination at 460 nm and 660 nm. We found that the loss of PSII activity, assayed by chlorophyll fluorescence, is faster than release of Mn from the Mn4CaO5 cluster, assayed by EPR. Loss of PSII activity and Mn release was slower during illumination in the presence of exogenous electron acceptors. Recovery of PSII activity was observed, after 30 min of addition of electron donor post illumination. The same behavior was observed under 460 and 660 nm illumination, suggesting stronger correlation between excessive excitation and photodamage compared to direct light absorption by the cluster. A unified model of PSII photodamage that takes into account present and previous literature reports is presented.

Hyperfine Sublevel Correlation Spectroscopy Studies of Iron-Sulfur Cluster in Rieske Protein from Green Sulfur Bacterium Chlorobaculum tepidum.

The magnetic properties of the Rieske protein purified from Chlorobaculum tepidum were investigated using electron paramagnetic resonance and hyperfine sublevel correlation spectroscopy (HYSCORE). The g-values of the Fe2S2 center were gx = 1.81, gy = 1.90, and gz = 2.03. Four classes of nitrogen signals were obtained by HYSCORE. Nitrogens 1 and 2 had relatively strong magnetic hyperfine couplings and were assigned as the nitrogen directly ligated to Fe. Nitrogens 3 and 4 had relatively weak magnetic hyperfine couplings and were assigned as the other nitrogen of the His ligands and peptide nitrogen connected to the sulfur atom via hydrogen bonding, respectively. The anisotropy of nitrogen 3 reflects the different spin density distributions on the His ligands, which influences the electron transfer to quinone.