Light-induced alteration of low-temperature interprotein electron transfer between photosystem I and flavodoxin

Electron paramagnetic resonance (EPR) was used to study light-induced electron transfer in Photosystem I-flavodoxin complexes. Deuteration of flavodoxin enables the signals of the reduced flavin acceptor and oxidized primary donor, P(700)(+), to be well-resolved at X- and D-band EPR. In dark-adapted samples, photoinitiated interprotein electron transfer does not occur at 5 K. However, for samples prepared in dim light, significant interprotein electron transfer occurs at 5 K and a concomitant loss of the spin-correlated radical pair P(+)A(1A)(-) signal is observed. These results indicate a light-induced reorientation of flavodoxin in the PSI docking site that allows a high quantum yield efficiency for the interprotein electron transfer reaction.     

Bidirectional electron transfer in photosystem I: electron transfer on the PsaA side is not essential for phototrophic growth in Chlamydomonas

We have used pulsed electron paramagnetic resonance (EPR) measurements of the electron spin polarised (ESP) signals arising from the geminate radical pair P700(z.rad;+)/A(1)(z.rad;-) to detect electron transfer on both the PsaA and PsaB branches of redox cofactors in the photosystem I (PSI) reaction centre of Chlamydomonas reinhardtii. We have also used electron nuclear double resonance (ENDOR) spectroscopy to monitor the electronic structure of the bound phyllosemiquinones on both the PsaA and PsaB polypeptides. Both these spectroscopic assays have been used to analyse the effects of site-directed mutations to the axial ligands of the primary chlorophyll electron acceptor(s) A(0) and the conserved tryptophan in the PsaB phylloquinone (A(1)) binding pocket. Substitution of histidine for the axial ligand methionine on the PsaA branch (PsaA-M684H) blocks electron transfer to the PsaA-branch phylloquinone, and blocks photoaccumulation of the PsaA-branch phyllosemiquinone. However, this does not prevent photoautotrophic growth, indicating that electron transfer via the PsaB branch must take place and is alone sufficient to support growth. The corresponding substitution on the PsaB branch (PsaB-M664H) blocks kinetic electron transfer to the PsaB phylloquinone at 100 K, but does not block the photoaccumulation of the phyllosemiquinone. This transformant is unable to grow photoautotrophically although PsaA-branch electron transfer to and from the phyllosemiquinone is functional, indicating that the B branch of electron transfer may be essential for photoautotrophic growth. Mutation of the conserved tryptophan PsaB-W673 to leucine affects the electronic structure of the PsaB phyllosemiquinone, and also prevents photoautotrophic growth.     

Semiquinone and ascorbyl radicals in the gut fluids of caterpillars measured with EPR spectrometry

The biological activity of phenolic compounds ingested by caterpillars is commonly believed to result from their oxidation, although the products of oxidation have been well-characterized in only a few cases. The initial oxidation products of phenols (semiquinone or phenoxyl radicals) can be measured with electron paramagnetic resonance (EPR) spectrometry. In this study semiquinone radicals formed from tannic acid and gallic acid in the gut fluids of two species of caterpillars were measured. In Orgyia leucostigma, in which ingested phenols are not oxidized, semiquinone radicals were absent or at very low intensities. By contrast, in Malacosoma disstria, in which ingested phenols are oxidized, high semiquinone radical intensities were measured. In the absence of detectable levels of semiquinone radicals, ascorbyl radicals were detected in the EPR spectra instead. High molar ratios of ascorbate to phenols in an artificial diet produced ascorbyl radicals in the midgut fluids of both species, while diets containing low molar ratios produced semiquinone radicals. Similar results were obtained in M. disstria fed the leaves of red oak or sugar maple. The results of this study provide further evidence that ascorbate is an essential antioxidant that prevents the oxidation of phenols in the gut fluids of caterpillars, and demonstrate that EPR spectrometry is a valuable method for determining the degree of oxidative activation of phenols ingested by herbivorous insects.     

Assembly of photosystem I. II. Rubredoxin is required for the in vivo assembly of F(X) in Synechococcus sp. PCC 7002 as shown by optical and EPR spectroscopy

The rubA gene was insertionally inactivated in Synechococcus sp. PCC 7002, and the properties of photosystem I complexes were characterized spectroscopically. X-band EPR spectroscopy at low temperature shows that the three terminal iron-sulfur clusters, F(X), F(A), and F(B), are missing in whole cells, thylakoids, and photosystem (PS) I complexes of the rubA mutant. The flash-induced decay kinetics of both P700(+) in the visible and A(1)- in the near-UV show that charge recombination occurs between P700(+) and A(1)- in both thylakoids and PS I complexes. The spin-polarized EPR signal at room temperature from PS I complexes also indicates that forward electron transfer does not occur beyond A(1). In agreement, the spin-polarized X-band EPR spectrum of P700(+) A(1)- at low temperature shows that an electron cycle between A(1)- and P700(+) occurs in a much larger fraction of PS I complexes than in the wild-type, wherein a relatively large fraction of the electrons promoted are irreversibly transferred to [F(A)/F(B)]. The electron spin polarization pattern shows that the orientation of phylloquinone in the PS I complexes is identical to that of the wild type, and out-of-phase, spin-echo modulation spectroscopy shows the same P700(+) to A(1)- center-to-center distance in photosystem I complexes of wild type and the rubA mutant. In contrast to the loss of F(X), F(B), and F(A), the Rieske iron-sulfur protein and the non-heme iron in photosystem II are intact. It is proposed that rubredoxin is specifically required for the assembly of the F(X) iron-sulfur cluster but that F(X) is not required for the biosynthesis of trimeric P700-A(1) cores. Since the PsaC protein requires the presence of F(X) for binding, the absence of F(A) and F(B) may be an indirect result of the absence of F(X).     

Electrostatic influence of PsaC protein binding to the PsaA/PsaB heterodimer in photosystem I

The absence of the PsaC subunit in the photosystem I (PSI) complex (native PSI complex) by mutagenesis or chemical manipulation yields a PSI core (P700-F(X) core) that also lacks subunits PsaD and PsaE and the two iron-sulfur clusters F(A) and F(B), which constitute an integral part of PsaC. In this P700-F(X) core, the redox potentials (E(m)) of the two quinones A(1A/B) and the iron-sulfur cluster F(X) as well as the corresponding protonation patterns are investigated by evaluating the electrostatic energies from the solution of the linearized Poisson-Boltzmann equation. The B-side specific Asp-B558 changes its protonation state significantly upon isolating the P700-F(X) core, being mainly protonated in the native PSI complex but ionized in the P700-F(X) core. In the P700-F(X) core, E(m)(A(1A/B)) remains practically unchanged, whereas E(m)(F(X)) is upshifted by 42 mV. With these calculated E(m) values, the electron transfer rate from A(1) to F(X) in the P700-F(X) core is estimated to be slightly faster on the A(1A) side than that of the wild type, which is consistent with kinetic measurements.     

Redox cycles of caffeic acid, alpha-tocopherol, and ascorbate: implications for protection of low-density lipoproteins against oxidation

This study addresses the dynamic interactions among alpha-tocopherol, caffeic acid, and ascorbate in terms of a sequence of redox cycles aimed at accomplishing optimal synergistic antioxidant protection. Several experimental models were designed to examine these interactions: UV irradiation of alpha-tocopherol-containing sodium dodecyl sulfate micelles, one-electron oxidations catalyzed by the hypervalent state of myoglobin, ferrylmyoglobin, and autoxidation at appropriate pHs. These models were assessed by ultraviolet (UV) and electron paramagnetic resonance (EPR), entailing direct- and continuous-flow experiments, spectroscopy and by separation and identification of products by HPLC. The alpha-tocopheroxyl radical EPR signal generated by UV irradiation of alpha-tocopherol-containing micelles was suppressed by caffeic acid and ascorbate; in the former case, no other EPR signal was observed at pH 7.4, whereas in the latter case, the alpha-tocopheroxyl radical EPR signal was replaced by a doublet EPR spectrum corresponding to the ascorbyl radical (A*-). The potential interactions between caffeic acid and ascorbate were further analyzed by assessing, on the one hand, the ability of ascorbate to reduce the caffeic acid o-semiquinone (generated by oxidation of caffeic acid by ferrylmyoglobin) and, on the other hand, the ability of caffeic acid to reduce ascorbyl radical (generated by autoxidation or oxidation of ascorbate by ferrylmyoglobin). The data presented indicate that the reductive decay of ascorbyl radical (A*-) and caffeic acid o-semiquinone (Caf-O*) can be accomplished by caffeic acid (Caf-OH) and ascorbate (AH-), respectively, thus pointing to the reversibility of the reaction Caf-O* + AH- <--> Caf-OH + A*-. Continuous-flow EPR measurements of mixtures containing ferrylmyoglobin, alpha-tocopherol-containing micelles, caffeic acid, and ascorbate revealed that ascorbate is the ultimate electron donor in the sequence encompassing transfer of the radical character from the micellar phase to the phase. In independent experiments, the effects of caffeic acid and ascorbate on the oxidation of two low-density lipoprotein (LDL) populations, control and alpha-tocopherol-enriched, were studied and results indicated that alpha-tocopherol, caffeic acid, and ascorbate acted synergistically to afford optimal protection of LDL against oxidation. These results are analyzed for each individual antioxidant in terms of three domains: its localization and that of the antioxidant-derived radical, its reduction potential, and the predominant decay pathways for the antioxidant-derived radical, that exert kinetic control on the process.     

Light-induced ascorbate-dependent electron transport and membrane energization in chloroplasts of bundle sheath cells of the C4 plant maize

Chloroplasts in bundle sheath cells (BSC) of maize perform photosystem I (PSI)-mediated production of ATP. In this study, the participation of ascorbate (Asc) as an electron donor to PSI in light-induced electron transport in isolated maize BSC was demonstrated. It was found that Asc, at physiological concentrations, rapidly reduced photooxidized reaction center chlorophyll of PSI (P700). The rate of Asc donation of electrons to P700+ reached rates of 50-100 microequivalents (mg Chl)(-1) h(-1) at 70-80 mM ascorbate with methyl viologen as an electron acceptor. Electron transport supported by Asc was coupled with membrane energization, as demonstrated by the light-induced formation of a trans-thylakoid electric field measured by the electrochromic shift of carotenoids. The possible physiological function of Asc-dependent electron transport in bundle sheath chloroplasts of maize, as an electron donor for linear electron flow versus sustaining cyclic electron transport, is discussed.     

Contribution of vitamin K1 to the electron spin polarization in spinach photosystem I

The electron spin polarized (ESP) electron paramagnetic resonance (EPR) signal observed in spinach photosystem I (PSI) particles was examined in preparations depleted of vitamin K1 by solvent extraction and following biological reconstitution by the quinone. The ESP EPR signal was not detected in the solvent-extracted PSI sample but was restored upon reconstitution with either protonated or deuterated vitamin K1 under conditions that also restored electron transfer to the terminal PSI acceptors. Reconstitution using deuterated vitamin K1 resulted in a line narrowing of the ESP EPR signal, supporting the conclusion that the ESP EPR signals in the reconstituted samples arise from a radical pair consisting of the oxidized PSI primary donor, P700+, and reduced vitamin K1.     

The reduction of ferrate(VI) to ferrate(V) by ascorbate

The reduction of ferrate(VI) by ascorbate has been studied under anaerobic conditions in the pH range between 6.8 and 11.5 at 24 degrees C. A mechanism is proposed that is consistent with the observed rate constants k11 (HFeO4- + AH-) = (5.6 +/- 0.6) x 10(6) M-1 s-1, k12(FeO4(2-) + AH-) = (1.3 +/- 0.1) x 10(6) M-1 s-1 and the pK(HFeO4- in equilibrium with H(+) + FeO4(2-) = 7.9. Stoichiometric studies show that at high ratios of [AH-]/[FeO4(2-)], one ferrate(VI) oxidizes three molecules of ascorbate to the corresponding ascorbyl (A-) radicals.     

A kinetic assessment of the sequence of electron transfer from F(X) to F(A) and further to F(B) in photosystem I: the value of the equilibrium constant between F(X) and F(A)

The x-ray structure analysis of photosystem I (PS I) crystals at 4-A resolution (Schubert et al., 1997, J. Mol. Biol. 272:741-769) has revealed the distances between the three iron-sulfur clusters, labeled F(X), F(1), and F(2), which function on the acceptor side of PS I. There is a general consensus concerning the assignment of the F(X) cluster, which is bound to the PsaA and PsaB polypeptides that constitute the PS I core heterodimer. However, the correspondence between the acceptors labeled F(1) and F(2) on the electron density map and the F(A) and F(B) clusters defined by electron paramagnetic resonance (EPR) spectroscopy remains controversial. Two recent studies (Diaz-Quintana et al., 1998, Biochemistry. 37:3429-3439;, Vassiliev et al., 1998, Biophys. J. 74:2029-2035) provided evidence that F(A) is the cluster proximal to F(X), and F(B) is the cluster that donates electrons to ferredoxin. In this work, we provide a kinetic argument to support this assignment by estimating the rates of electron transfer between the iron-sulfur clusters F(X), F(A), and F(B). The experimentally determined kinetics of P700(+) dark relaxation in PS I complexes (both F(A) and F(B) are present), HgCl(2)-treated PS I complexes (devoid of F(B)), and P700-F(X) cores (devoid of both F(A) and F(B)) from Synechococcus sp. PCC 6301 are compared with the expected dependencies on the rate of electron transfer, based on the x-ray distances between the cofactors. The analysis, which takes into consideration the asymmetrical position of iron-sulfur clusters F(1) and F(2) relative to F(X), supports the F(X) --> F(A) --> F(B) --> Fd sequence of electron transfer on the acceptor side of PS I. Based on this sequence of electron transfer and on the observed kinetics of P700(+) reduction and F(X)(-) oxidation, we estimate the equilibrium constant of electron transfer between F(X) and F(A) at room temperature to be approximately 47. The value of this equilibrium constant is discussed in the context of the midpoint potentials of F(X) and F(A), as determined by low-temperature EPR spectroscopy.     

FTIR study of the primary electron donor of photosystem I (P700) revealing delocalization of the charge in P700(+) and localization of the triplet character in (3)P700.

The effect of global (15)N or (2)H labeling on the light-induced P700(+)/P700 FTIR difference spectra has been investigated in photosystem I samples from Synechocystis at 90 K. The small isotope-induced frequency shifts of the carbonyl modes observed in the P700(+)/P700 spectra are compared to those of isolated chlorophyll a. This comparison shows that bands at 1749 and 1733 cm(-)(1) and at 1697 and 1637 cm(-)(1), which upshift upon formation of P700(+), are candidates for the 10a-ester and 9-keto C=O groups of P700, respectively. A broad and relatively weak band peaking at 3300 cm(-)(1), which does not shift upon global labeling or (1)H-(2)H exchange, is ascribed to an electronic transition of P700(+), indicating that at least two chlorophyll a molecules (denoted P(1) and P(2)) participate in P700(+). Comparisons of the (3)P700/P700 FTIR difference spectrum at 90 K with spectra of triplet formation in isolated chlorophyll a or in RCs from photosystem II or purple bacteria identify the bands at 1733 and 1637 cm(-)(1), which downshift upon formation of (3)P700, as the 10a-ester and 9-keto C=O modes, respectively, of the half of P700 that bears the triplet (P(1)). Thus, while the P(2) carbonyls are free from interaction, both the 10a-ester and the 9-keto C=O of P(1) are hydrogen bonded and the latter group is drastically perturbed compared to chlorophyll a in solution. The Mg atoms of P(1) and P(2) appear to be five-coordinated. No localization of the triplet on the P(2) half of P700 is observed in the temperature range of 90-200 K. Upon P700 photooxidation, the 9-keto C=O bands of P(1) and P(2) upshift by almost the same amount, giving rise to the 1656(+)/1637(-) and 1717(+)/1697(-) cm(-)(1) differential signals, respectively. The relative amplitudes of these differential signals, as well as of those of the 10a-ester C=O modes, appear to be slightly dependent on sample orientation and temperature and on the organism used to generate the P700(+)/P700 spectrum. If it is assumed that the charge density on ring V of chlorophyll a, as measured by the perturbation of the 10a-ester or 9-keto C=O IR vibrations, mainly reflects the spin density on the two halves of the oxidized P700 special pair, a charge distribution ranging from 1:1 to 2:1 (in favor of P(2)) is deduced from the measurements presented here. The extreme downshift of the 9-keto C=O group of P(1), indicative of an unusually strong hydrogen bond, is discussed in relation with the models previously proposed for the PSI special pair.     

Low-temperature interquinone electron transfer in photosynthetic reaction centers from Rhodobacter sphaeroides and Blastochloris viridis: characterization of Q(B)- states by high-frequency electron paramagnetic resonance (EPR) and electron-nuclear double resonance (ENDOR)

High-frequency electron paramagnetic resonance (HF EPR) techniques have been employed to look for localized light-induced conformational changes in the protein environments around the reduced secondary quinone acceptor (Q(B)(-)) in Rhodobacter sphaeroides and Blastochloris viridis RCs. The Q(A)(-) and Q(B)(-) radical species in Fe-removed/Zn-replaced protonated RCs substituted with deuterated quinones are distinguishable with pulsed D-band (130 GHz) EPR and provide native probes of both the low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron-transfer event and the structure of trapped conformational substates. We report here the first spectroscopic evidence that cryogenically trapped, light-induced changes enable low-temperature Q(A)(-)Q(B) --> Q(A)Q(B)(-) electron transfer in the B. viridis RC and the first observation of an inactive, trapped P(+)Q(B)(-) state in both R. sphaeroides and B. viridis RCs that does not recombine at 20 K. The high resolution and orientational selectivity of HF electron-nuclear double resonance (ENDOR) allows us to directly probe protein environments around Q(B)(-) for distinct P(+)Q(B)(-) kinetic RC states by spectrally selecting specific nuclei in isotopically labeled samples. No structural differences in the protein structure near Q(B)(-) or reorientation (within 5 degrees ) of Q(B)(-) was observed with HF ENDOR spectra of two states of P(+)Q(B)(-): "active" and "inactive" states with regards to low-temperature electron transfer. These results reveal a remarkably enforced local protein environment for Q(B) in its reduced semiquinone state and suggest that the conformational change that controls reactivity resides beyond the Q(B) local environment.     

Inhibition of photosystems I and II and enhanced back flow of photosystem I electrons in cucumber leaf discs chilled in the light

Pre-illumination of cucumber leaf discs at a chilling temperature in low-irradiance white light resulted in accelerated re-reduction of P700(+) [the special Chl pair in the photosystem I (PSI) reaction centre] when the far-red measuring light was turned off. Measurements (in +/- methyl viologen or +/- DCMU conditions) of the re-reduction half time suggest that accelerated re-reduction of P700(+) appeared to be predominantly due to charge recombination and only partly due to reductants sustained by previous cyclic electron flow around PSI. Apparently, charge recombination in PSI was greatly enhanced by inhibition of forward, linear electron flow. Inhibition of PSII electron transport was observed to occur to a lesser extent than that of PSI, but only if the measurement of PSII functionality was free from complications due to downstream accumulation of electrons in pools. We suggest that promotion of controlled charge recombination and cyclic electron flow round PSI during chilling of leaves in the light may partly prevent further damage to both photosystems.     

ESR signal of the iron-sulfur center F(X) and its function in the homodimeric reaction center of Heliobacterium modesticaldum

Electron transfer in the membranes and the type I reaction center (RC) core protein complex isolated from Heliobacterium modesticaldum was studied by optical and ESR spectroscopy. The RC is a homodimer of PshA proteins. In the isolated membranes, illumination at 14 K led to accumulation of a stable ESR signal of the reduced iron-sulfur center F(B)(-) in the presence of dithiothreitol, and an additional 20 min illumination at 230 K induced the spin-interacting F(A)(-)/F(B)(-) signal at 14 K. During illumination at 5 K in the presence of dithionite, we detected a new transient signal with the following values: g(z)= 2.040, g(y)= 1.911, and g(x)= 1.896. The signal decayed rapidly with a 10 ms time constant after the flash excitation at 5 K and was attributed to the F(X)(-)-type center, although the signal shape was more symmetrical than that of F(X)(-) in photosystem I. In the purified RC core protein, laser excitation induced the absorption change of a special pair, P800. The flash-induced P800(+) signal recovered with a fast 2-5 ms time constant below 150 K, suggesting charge recombination with F(X)(-). Partial destruction of the RC core protein complex by a brief exposure to air increased the level of the P800(+)A(0)(-) state that gave a lifetime (t(1/2)) of 100 ns at 77 K. The reactions of F(X) and quinone were discussed on the basis of the three-dimensional structural model of RC that predicts the conserved F(X)-binding site and the quinone-binding site, which is more hydrophilic than that in the photosystem I RC.     

Spin trapping endogenous radicals in MC-1010 cells: Evidence for hydroxyl radical and carbon-centered ascorbyl radical adducts

Incubation of MC-1010 cells with the spin-trapping agent 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) followed by brief treatment with the solid oxidant lead dioxide (PbO₂) yielded, after filtration, a cell-free solution that contained two nitroxyl adducts. The first was the hydroxyl radical adduct, 5,5-dimethyl-2-hydroxypyrrolidine-1-oxyl (DMPO-OH), which formed immediately upon PbO₂ oxidation. The second had a 6-line EPR spectrum typical of a carbon-centered radical (A_N=15.9 G; A_H=22.4 G) and formed more slowly. No radical signals were detected in the absence of either cells or PbO₂ treatment. The 6-line spectrum could be duplicated in model systems that contained ascorbate, DMPO and DMPO-OH, where the latter was formed from hydroxyl radicals generated by sonolysis or the cleavage of hydrogen peroxide with Fe²⁺ (Fenton reaction). In addition, enrichment of MC-1010 cells with ascorbate prior to spin trapping yielded the 6-line EPR spectrum as the principal adduct following PbO₂ oxidation and filtration. These results suggest that ascorbate reacted with DMPO-OH to form a carbon-centered ascorbyl radical that was subsequently trapped by DMPO. The requirement for mild oxidation to detect the hydroxyl radical adduct suggests that DMPO-OH formed in the cells was reduced to an EPR-silent form (i.e., the hydroxylamine derivative). Alternatively, the hydroxylamine derivative was the species initially formed. The evidence for endogenous hydroxyl radical formation in unstimulated leukocytes may be relevant to the leukemic nature of the MC-1010 cell line. The spin trapping of the ascorbyl radical is the first report of formation of the carbon-centered ascorbyl radical by means other than pulse radiolysis. Unless it is spin trapped, the carbon-centered ascorbyl radical immediately rearranges to the more stable oxygen-centered species that is passive to spin trapping and characterized by the well-known EPR doublet of A_H4=1.8 G.Abbreviation EPR Electron Paramagnetic Resonance     

Quantification of photosystem I and II in different parts of the thylakoid membrane from spinach

Electron paramagnetic resonance (EPR) was used to quantify Photosystem I (PSI) and PSII in vesicles originating from a series of well-defined but different domains of the thylakoid membrane in spinach prepared by non-detergent techniques. Thylakoids from spinach were fragmented by sonication and separated by aqueous polymer two-phase partitioning into vesicles originating from grana and stroma lamellae. The grana vesicles were further sonicated and separated into two vesicle preparations originating from the grana margins and the appressed domains of grana (the grana core), respectively. PSI and PSII were determined in the same samples from the maximal size of the EPR signal from P700(+) and Y(D)( .-), respectively. The following PSI/PSII ratios were found: thylakoids, 1.13; grana vesicles, 0.43; grana core, 0.25; grana margins, 1.28; stroma lamellae 3.10. In a sub-fraction of the stroma lamellae, denoted Y-100, PSI was highly enriched and the PSI/PSII ratio was 13. The antenna size of the respective photosystems was calculated from the experimental data and the assumption that a PSII center in the stroma lamellae (PSIIbeta) has an antenna size of 100 Chl. This gave the following results: PSI in grana margins (PSIalpha) 300, PSI (PSIbeta) in stroma lamellae 214, PSII in grana core (PSIIalpha) 280. The results suggest that PSI in grana margins have two additional light-harvesting complex II (LHCII) trimers per reaction center compared to PSI in stroma lamellae, and that PSII in grana has four LHCII trimers per monomer compared to PSII in stroma lamellae. Calculation of the total chlorophyll associated with PSI and PSII, respectively, suggests that more chlorophyll (about 10%) is associated with PSI than with PSII.     

Radical formation in cytochrome c oxidase

The formation of radicals in bovine cytochrome c oxidase (bCcO), during the O(2) redox chemistry and proton translocation, is an unresolved controversial issue. To determine if radicals are formed in the catalytic reaction of bCcO under single turnover conditions, the reaction of O(2) with the enzyme, reduced by either ascorbate or dithionite, was initiated in a custom-built rapid freeze quenching (RFQ) device and the products were trapped at 77K at reaction times ranging from 50μs to 6ms. Additional samples were hand mixed to attain multiple turnover conditions and quenched with a reaction time of minutes. X-band (9GHz) continuous wave electron paramagnetic resonance (CW-EPR) spectra of the reaction products revealed the formation of a narrow radical with both reductants. D-band (130GHz) pulsed EPR spectra allowed for the determination of the g-tensor principal values and revealed that when ascorbate was used as the reductant the dominant radical species was localized on the ascorbyl moiety, and when dithionite was used as the reductant the radical was the SO(2)(-) ion. When the contributions from the reductants are subtracted from the spectra, no evidence for a protein-based radical could be found in the reaction of O(2) with reduced bCcO. As a surrogate for radicals formed on reaction intermediates, the reaction of hydrogen peroxide (H(2)O(2)) with oxidized bCcO was studied at pH 6 and pH 8 by trapping the products at 50μs with the RFQ device to determine the initial reaction events. For comparison, radicals formed after several minutes of incubation were also examined, and X-band and D-band analysis led to the identification of radicals on Tyr-244 and Tyr-129. In the RFQ measurements, a peroxyl (ROO) species was formed, presumably by the reaction between O(2) and an amino acid-based radical. It is postulated that Tyr-129 may play a central role as a proton loading site during proton translocation by ejecting a proton upon formation of the radical species and then becoming reprotonated during its reduction via a chain of three water molecules originating from the region of the propionate groups of heme a(3). This article is part of a Special Issue entitled: "Allosteric cooperativity in respiratory proteins".     

The two histidine axial ligands of the primary electron donor chlorophylls (P700) in photosystem I are similarly perturbed upon P700+ formation

The extent of delocalization of the positive charge in the oxidized dimer of chlorophyll (Chl) constituting P700, the primary electron donor of photosystem I (PSI), has been investigated by analyzing the perturbation upon P700(+) formation of infrared (IR) vibrational modes of the two His axial ligands of the two P700 Chl molecules. Fourier transform IR (FTIR) difference spectra of the photooxidation of P700 in PSI core complexes isolated from Synechocystis sp. PCC 6803 isotopically labeled either globally with (15)N or more specifically with (13)C on all the His residues reveal isotopic shifts of a differential signal at 1102/1108 cm(-)(1). This signal is assigned to a downshift upon P700(+) formation of the predominantly C(5)-Ntau imidazole stretching mode of His residue(s). The amplitude of this signal is reduced by approximately half in FTIR spectra of Synechocystis mutants in which His PsaB 651, the axial ligand to one of the two Chl molecules in P700, is replaced by Cys, Gln, or Leu. These observations provide further evidence that the positive charge in P700(+) is essentially delocalized over the two Chl molecules, in agreement with a previous FTIR study in which the frequency of the vibrational modes of the 9-keto and 10a-ester C=O groups of the two Chl's in P700, P700(+), and (3)P700 were firmly established for the first time [Breton, J., et al. (1999) Biochemistry 38, 11585-11592]. Only limited perturbations of the amplitude and frequency of the 9-keto and 10a-ester C=O bands of the P700 Chl are elicited by the mutations. On the basis of comparable mutational studies of the primary electron donor in purple bacteria, these perturbations are attributed to small molecular rearrangements of the Chl macrocycle and substituents caused by the repositioning of the P700 dimer in the new protein cavity generated by the mutations. It is proposed that the perturbation of the FTIR spectra upon mutation of a His axial ligand of the P700 Chl recently reported in Chlamydomonas reinhardtii [Hastings, G., et al. (2001) Biochemistry 40, 12943-12949] can be explained by the same effect without the need for a new assignment of the C=O bands of P700. The distribution of charge/spin in P700(+) and (3)P700 determined by FTIR spectroscopy is discussed in relation with the contrasting interpretations derived from recent magnetic resonance experiments.     

Ascorbyl free radical as a reliable indicator of free-radical-mediated myocardial ischemic and post-ischemic injury. A real-time continuous-flow ESR study

The real-time kinetics of the release of ascorbyl free radicals in the coronary perfusate from isolated rat hearts submitted to an ischemia/reperfusion sequence has been achieved by continuous-flow ESR using high-speed acquisition techniques. Enhanced ESR detection of ascorbyl free radicals was obtained by addition of dimethyl sulfoxide (Me2SO), a strong cation chelator and oxidizing agent. A continuous-flow device allowed a direct monitoring of the ascorbyl free radical and/or ascorbate leakage in coronary perfusate by observation of the ascorbyl radical doublet (aH = 0.188 mT and g = 2.0054). 1. The results showed that ascorbyl free radical release occurred mainly during sequences of low-flow ischemia (90 min) coupled or not with 30 min of zero-flow ischemia followed by reperfusion (60 min). The kinetic profiles of ascorbyl-free-radical detection confirm in quantitative terms the expected correlation between the duration of the ischemic insult and the magnitude of ascorbate extracellular release upon reperfusion. There is indication that ascorbyl free radical depletion could be secondary to oxygen-derived-free-radical-induced cellular damage. 2. The amount of residual ascorbic acid was quantitated on myocardial tissue at the end of reperfusion using Me2SO as extracting solvent. Intense oxidation of ascorbate and chemical stabilization of the resulting free radical species provided by Me2SO allowed ESR measurement of a marked tissue ascorbate depletion related to the duration of ischemia. 3. Perfusion of superoxide dismutase during low-flow ischemia and the first 10 min of reperfusion greatly inhibited both extracellular release and endogenous ascorbate depletion. These results suggest that the ascorbate redox system constitutes a major protective mechanism against free-radical-induced myocardial injury. 4. The proposed direct ESR detection of ascorbyl free radicals in the coronary perfusates or in tissue extracts does not require extensive chemical preparation and conditioning of effluent or tissue samples. It provides an interesting straightforward alternative to the evaluation of detrimental free radical processes affecting the myocardium during ischemia and reperfusion.     

Effects of Dark Adaptation on Light-Induced Electron Transport through Photosystem I in the Cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Kinetics of the redox reactions in the reaction center (P700) of photosystem I (PSI) of the cyanobacterium Synechocystis sp. PCC 6803 have been studied by EPR spectroscopy. The redox kinetics were recorded based on accumulation of the EPRI signal when the final signal was the sum of individual signals produced in response to illumination of the cells. After prolonged (more than 3 sec) dark intervals between illuminations, the kinetic curve of the EPR signal from P700+ was multiphasic. After a sharp increase in the signal amplitude at the beginning of illumination (phase I), the amplitude rapidly (for 0.1-0.2 sec) decreased (phase II). Then the signal amplitude gradually increased (phase III) until the steady rate of electron transfer was established. With short-term (1 sec) dark intervals between the flashes and also in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the kinetics of the light-induced increase in the EPR signal from P700+ were monophasic. Inhibition with iodoacetamide of electron transport on the acceptor side of PSI under anaerobic conditions or an increase in the amount of respiration substrates on addition of glucose into a suspension of DCMU-treated wild-type cells increased the level of P700 reduction in phase III. The findings suggest that the kinetic curve of the EPR signal from P700+ is determined by both the electron entrance onto P700+ on the donor side of PSI and activity of electron acceptors of PSI.