Exploring fast electron transfer processes by magnetic fields.

Photoinduced electron transfer generates radical pairs which recombine with 10(-9)10(-8)s by electron back-transfer to either singlet or triplet products. The product distribution determined by the spin motion of the unpaired electrons in the radical pairs is affected by external magnetic fields. The analysis of the magnetic field effect furnishes new information about electron transfer processes. Light-induced electron transfer in polar solvents and in the bacterial photosynthetic reaction center are discussed as examples.     

Biradicals by triplet recombination of radical ion pairs.

Radical ion pairs generated by photoinduced electron transfer may undergo return electron transfer (RET) in pairs of singlet or triplet multiplicity. RET efficiencies are determined by the free energy of RET and the topologies of the potential surfaces of parent molecule, radical ion and triplet state. If radical ion geometries are different from the corresponding triplet states, RET occurs either with cleavage ("dissociative" RET; 1,2-diphenylcyclopropane radical cations) or formation of C-C bonds ("associative" RET; norbornadiene radical cation). Radical ions of some strained ring compounds spontaneously undergo ring-opening; RET to such species form ring-opened triplets without major geometry changes. CIDNP spectroscopy offers unique insights into triplet RET.     

Radical cations and triplet states of 1,2-disubstituted cyclopropanes: comparison of potential surfaces

Radical ion pairs generated by photo-induced electron transfer from 1,2-disubstituted cyclopropanes to various acceptors undergo return electron transfer in pairs of singlet and triplet multiplicity. The pair energies relative to the reactant ground states and to accessible triplet states, respectively, determine the competition between the recombination pathways. The potential surfaces of the radical cations and triplet states of 1,2-diphenyl-, 1, and 1,2-dimethylcyclopropane, 2, have been examined by density functional theory calculations. The radical cation surfaces have minima at geometries that retain significant bonding between C-1 and C-2, preventing geometric isomerization of the radical cations. The triplet potential surfaces are dissociative with minimal rotational differentiation at long distances between C-1 and C-2.     

EPR characterisation of the triplet state in photosystem II reaction centers with singly reduced primary acceptor Q(A)

The triplet states of photosystem II core particles from spinach were studied using time-resolved cw EPR technique at different reduction states of the iron--quinone complex of the reaction center primary electron acceptor. With doubly reduced primary acceptor, the well-known photosystem II triplet state characterised by zero-field splitting parameters |D|=0.0286 cm(-1), |E|=0.0044 cm(-1) was detected. When the primary acceptor was singly reduced either chemically or photochemically, a triplet state of a different spectral shape was observed, bearing the same D and E values and characteristic spin polarization pattern arising from RC radical pair recombination. The latter triplet state was strongly temperature dependent disappearing at T=100 K, and had a much faster decay than the former one. Based on its properties, this triplet state was also ascribed to the photosystem II reaction center. A sequence of electron-transfer events in the reaction centers is proposed that explains the dependence of the triplet state properties on the reduction state of the iron--quinone primary acceptor complex.     

Triplet state of the primary donor in reaction centers of the phototrophic bacterium Rhodobacter sphaeroides R26 with active photoinduced electron transfer

EPR characteristics of transient paramagnetic states photoinduced in isolated reaction centers of Rhodobacter sphaeroides R26 with intact electron transfer have been studied. It was demonstrated that the detected weak triplet state EPR signal belongs to the primary donor molecule and is populated via the conventional mechanism of radical pair S-T0 mixing. The distortion of the spectral shape of this signal is explained by the triplet quantum yield anisotropy brought about by the short lifetime of precursor radical pairs. The angular dependence of the anisotropy was evaluated. It was shown that the spectral shape of the triplet state of photosystem II reaction center observed in the case of singly-reduced primary quinone acceptor can also be described by the anisotropic quantum yield of the triplet, with practically the same angular dependence. These properties confirm the conclusions on the mechanism of photoinduced electron transfer in photosystem II, made in previous publications. The peculiarities in the functioning of photosystem II reaction centers are probably determined by strict limitations on the triplet state generation.     

Physical and chemical properties of pyropheophorbide-a methyl ester in ethanol, phosphate buffer and aqueous dispersion of small unilamellar dimyristoyl-L-alpha-phosphatidylcholine vesicles.

The aggregation process of pyropheophorbide-a methyl ester (PPME), a second-generation photosensitizer, was investigated in various solvents. Absorption and fluorescence spectra showed that the photosensitizer was under a monomeric form in ethanol as well as in dimyristoyl-L-alpha-phosphatidylcholine liposomes while it was strongly aggregated in phosphate buffer. A quantitative determination of reactive oxygen species production by PPME in these solvents has been undertaken by electron spin resonance associated with spin trapping technique and absorption spectroscopy. In phosphate buffer, both electron spin resonance and absorption measurements led to the conclusion that singlet oxygen production was not detectable while hydroxyl radical production was very weak. In liposomes and ethanol, singlet oxygen and hydroxyl radical production increased highly; the singlet oxygen quantum yield was determined to be 0.2 in ethanol and 0.13 in liposomes. The hydroxyl radical production origin was also investigated. Singlet oxygen was formed from PPME triplet state deactivation in the presence of oxygen. Indeed, the triplet state formation quantum yield of PPME was found to be about 0.23 in ethanol, 0.15 in liposomes (too small to be measured in PBS).     

Triplet state of the primary donor in reaction centers of the phototrophic bacterium <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> R26 with active photoinduced electron transfer

EPR characteristics of transient paramagnetic states photoinduced in isolated reaction centers of Rhodobacter sphaeroides R26 with intact electron transfer have been studied. It was demonstrated that the detected weak triplet state EPR signal belongs to the primary donor molecule and is populated via the conventional mechanism of radical pair S-T₀ mixing. The distortion of the spectral shape of this signal is explained by the triplet quantum yield anisotropy brought about by the short lifetime of precursor radical pairs. The angular dependence of the anisotropy was evaluated. It was shown that the spectral shape of the triplet state of photosystem II reaction center observed in the case of singly-reduced primary quinone acceptor can also be described by the anisotropic quantum yield of the triplet, with practically the same angular dependence. These properties confirm the conclusions on the mechanism of photoinduced electron transfer in photosystem II, made in previous publications. The peculiarities in the functioning of photosystem II reaction centers are probably determined by strict limitations on the triplet state generation.     

A light-induced spin-polarized triplet detected by EPR in photosystem II reaction centers

A light-induced spin-polarized triplet state has been detected in a purified Photosystem II preparation by electron paramagnetic resonance spectroscopy at liquid helium temperature. The electron spin polarization pattern is interpreted to indicate that the triplet originates from radical pair recombination between the oxidized primary donor chlorophyll, P-680+, and the reduced intermediate pheophytin, I-, as has been previously demonstrated in bacterial reaction centers. The dependence of the triplet signal on the redox state of I and the primary acceptor, Q, are consistent with the origin of the triplet signal from the triplet state of P-680. Redox-poising experiments indicate the presence of an endogenous donor (or donors) which operates at 3-5 K and 200 K. The zero field-splitting parameters of the triplet are very similar to those of monomeric chlorophyll a however, this alone does not allow a distinction to be made between monomeric and dimeric structures for P-680.     

Mechanism of the phospholipid transfer protein-mediated transfer of phospholipids from model lipid vesicles to high density lipoproteins

To study the effects of the phospholipid transfer protein (PLTP) on the thermodynamic parameters governing the transfer of phospholipids (PL) from single bilayer vesicles (SBV) to high density lipoprotein (HDL), we performed transfer measurements at various temperatures between 4 and 65 degrees C, using a pyrenylphosphatidylcholine (Pyr-PC) as probe. The proportion of excimer (E) to monomer (M) fluorescence of a pyrenyl moiety constitutes a direct measure of its local concentration. The transfers of Pyr-PC were monitored by following the decrease of E/M. The data were used to calculate the rate constants K(+1) for the transfer from SBV to HDL and to generate the corresponding Arrhenius plots. The equilibrium constants, K(eq), for the same reactions were also determined and used to generate Van't Hoff plots. From these data, we calculated the thermodynamic parameters for both the whole transfer reaction and the transition state. Both K(+1) and K(eq) values clearly varied with temperature. PLTP induced very similar decreases in the free energy for the whole reaction (DeltaG) and in that for the transition state (DeltaG(#)). At 37 degrees C, the decreases were of 0.37 and 0.29 kcal/mol, respectively. We studied the thermal denaturation of PLTP between 37 and 65 degrees C, and the effects of denatured PLTP samples on the PL transfer reaction were then determined. In all cases, the changes of DeltaG remained comparable to those of DeltaG(#). Thus the essential action of PLTP is to facilitate the first step of the reaction, which can be considered as the desorption of PL molecules from the surface of donor particles.     

Fate of flavins in sensitized photodegradation of isohumulones and reduced derivatives: studies on formation of radicals via EPR combined with detailed product analyses.

Photodegradation of isohumulones accounts for formation of the lightstruck flavor in beer. The reactions involved are mediated by riboflavin, a natural photosensitizer present in beer in ppb quantities. The results of an investigation of this sensitized degradation process are presented herein. Product analyses and electron paramagnetic resonance spectroscopy, in steady-state as well as in time-resolved mode, offer extensive insight into the photophysical and photochemical details of the degradation mechanism. In contrast to energy transfer and Norrish type I alpha-cleavage reactions that take place on direct irradiation of isohumulones, the sensitization pathway proceeds via one-electron redox chemistry involving the excited triplet state of riboflavin and derivatives. The flavin semiquinone radical thus formed could be readily detected, either by steady state or by time-resolved electron paramagnetic resonance spectroscopy. Superimposed signals in the spectra revealed the presence of radical fragments derived from isohumulones or tetrahydroisohumulones, which, on recombination with riboflavin semiquinone radicals, produced stable reaction products that were identified by HPLC-MS. However, no superimposed signals were observed on sensitized irradiation of dihydroisohumulones.     

Spin-orbit couplings in the DNA base pairs

The radiative lifetimes of the phosphorescent states of the adenine.thymine (A.T) and guanine.cytosine (G.C) base pairs were calculated on the basis of the singlet-triplet transition probability induced by spin-orbit couplings. The calculated radiative lifetimes averaged over the triplet sublevels of spin state were in the order of G.C less than A.T and in good correlation with those of the composite bases. On the whole the results suggested an important role for thymine triplet having a relatively long lifetime during the course of the triplet localization in DNA, in agreement with the experimental observation that the concentration of triplet is remarkably enhanced with increase in A+T content.     

Pulsed EPR spectroscopy on short-lived intermediates in Photosystem I.

The application of pulsed electron paramagnetic resonance spectroscopy on short-lived intermediates in Photosystem I is reviewed. The spin polarization in light-induced radical pairs gives rise to a phase shifted 'out-of-phase' electron spin echo signal. This echo signal shows a prominent modulation of its intensity as a function of the spacing between the two microwave pulses. Its modulation frequency is determined by the electron-electron spin couplings within the radical pair. Thereby, the measurement of the dipolar coupling gives direct information about the spin-spin distance and can therefore be used to determine cofactor distances with high precision. Application of this technique to the radical pair P(*+)(700)A(*-)(1) in Photosystem I is discussed. Moreover, if oriented samples (e.g. single crystals) are used, the angular dependence of the dipolar coupling can be used to derive the orientation of the axis connecting donor and acceptor with respect to an external (crystal) axes system. Using out-of-phase electron spin echo envelope modulation spectroscopy, the localization of the secondary acceptor quinone A(1) has become possible.     

High thermal stability of 3-isopropylmalate dehydrogenase from Thermus thermophilus resulting from low DeltaC(p) of unfolding.

To characterize the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of the enzyme and of its mesophilic counterpart from Escherichia coli was investigated at various temperatures. The unfolding curves were analyzed with a three-state model for E.coli IPMDH and with a two-state model for T.thermophilus IPMDH, to obtain the free energy change DeltaG degrees of each unfolding process. Other thermodynamic parameters, enthalpy change DeltaH, entropy change DeltaS and heat capacity change DeltaC(p), were derived from the temperature dependence of DeltaG degrees. The main feature of the thermophilic enzyme was its lower dependence of DeltaG degrees on temperature resulting from a low DeltaC(p). The thermophilic IPMDH had a significantly lower DeltaC(p), 1.73 kcal/mol.K, than that of E.coli IPMDH (20.7 kcal/mol.K). The low DeltaC(p) of T.thermophilus IPMDH could not be predicted from its change in solvent-accessible surface area DeltaASA. The results suggested that there is a large structural difference between the unfolded state of T.thermophilus and that of E.coli IPMDH. Another responsible factor for the higher thermal stability of T.thermophilus IPMDH was the increase in the most stable temperature T(s). The DeltaG degrees maximum of T.thermophilus IPMDH was much smaller than that of E.coli IPMDH. The present results clearly demonstrated that a higher melting temperature T(m) is not necessarily accompanied by a higher DeltaG degrees maximum.     

Transient EPR: using spin polarization in sequential radical pairs to study electron transfer in photosynthesis

The light induced electron transfer in photosynthesis generates a series of sequential spin polarized radical pairs, and transient electron paramagnetic resonance (TREPR) is ideally suited to study the lifetimes and physical and electronic structures of these radical pairs. In this article, the basic principles of TREPR are outlined with emphasis on the electron spin polarization (ESP) that develops during the electron transfer process. Examples of the analysis of TREPR data are given to illustrate the information that can be obtained. Recent applications of the technique to study the functionality of reaction centers, light-induced structural changes, and protein–cofactor interactions are reviewed.     

Blue light perception in plants. Detection and characterization of a light-induced neutral flavin radical in a C450A mutant of phototropin

The LOV2 domain of Avena sativa phototropin and its C450A mutant were expressed as recombinant fusion proteins and were examined by optical spectroscopy, electron paramagnetic resonance, and electron-nuclear double resonance. Upon irradiation (420-480 nm), the LOV2 C450A mutant protein gave an optical absorption spectrum characteristic of a flavin radical even in the absence of exogenous electron donors, thus demonstrating that the flavin mononucleotide (FMN) cofactor in its photogenerated triplet state is a potent oxidant for redox-active amino acid residues within the LOV2 domain. The FMN radical in the LOV2 C450A mutant is N(5)-protonated, suggesting that the local pH close to the FMN is acidic enough so that the cysteine residue in the wild-type protein is likely to be also protonated. An electron paramagnetic resonance analysis of the photogenerated FMN radical gave information on the geometrical and electronic structure and the environment of the FMN cofactor. The experimentally determined hyperfine couplings of the FMN radical point to a highly restricted delocalization of the unpaired electron spin in the isoalloxazine moiety. In the light of these results a possible radical-pair mechanism for the formation of the FMN-C(4a)-cysteinyl adduct in LOV domains is discussed.     

EPR characterisation of the triplet state in photosystem II reaction centers with singly reduced primary acceptor QA

The triplet states of photosystem II core particles from spinach were studied using time-resolved cw EPR technique at different reduction states of the iron-quinone complex of the reaction center primary electron acceptor. With doubly reduced primary acceptor, the well-known photosystem II triplet state characterised by zero-field splitting parameters |D| = 0.0286 cm⁻¹, |E| = 0.0044 cm⁻¹ was detected. When the primary acceptor was singly reduced either chemically or photochemically, a triplet state of a different spectral shape was observed, bearing the same D and E values and characteristic spin polarization pattern arising from RC radical pair recombination. The latter triplet state was strongly temperature dependent disappearing at T = 100 K, and had a much faster decay than the former one. Based on its properties, this triplet state was also ascribed to the photosystem II reaction center. A sequence of electron-transfer events in the reaction centers is proposed that explains the dependence of the triplet state properties on the reduction state of the iron-quinone primary acceptor complex.     

Characterization of the transient species generated by the photoexcitation of C-phycocyanin from Spirulina platensis: a laser photolysis and pulse radiolysis study

Nanosecond laser flash photolysis and pulse radiolysis were used to generate and characterize the triplet state and cation radical of C-phycocyanin (C-PC) from Spirulina platensis. The transient absorption spectra of C-PC were measured from direct excitation and acetone sensitization in aqueous solution at room temperature by KrF (248 nm) laser flash photolysis. Laser-induced transient species have been characterized by the method of acetone sensitization and one-electron oxidation. In nitrous oxide-saturated phosphate buffer saline (pH = 7.0) of C-PC, the produced intermediates are assigned to the excited triplet state and the radical cation. Using acetone as photosensitizer, the C-PC excited triplet states produced via triplet-triplet energy transfer and the C-PC radical cation from electron transfer reaction were further confirmed. Furthermore, the corresponding kinetic parameters were determined. To our knowledge, the transient absorption spectra of C-PC have been reported for the first time.     

Interaction between TOAC free radical and photoexcited triplet chromophores linked to peptide templates

The intramolecular quenching of photoexcited triplet states by free radicals linked to peptide templates was studied by time-resolved electron paramagnetic resonance (EPR) with pulsed laser excitation. The systems investigated are 3(10)-helix forming peptides, having in the amino acid sequence the free radical 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid (TOAC) and a triplet precursor, such as Bin, Bpa, or Trp, incorporated at different relative positions. Upon interaction with the excited triplet the TOAC radical spin sublevel populations assume values that differ from the Boltzmann equilibrium values. This spin polarization effect produces EPR lines in emission whose time evolution reflects the triplet quenching rate. In particular, in a series of peptides labeled with Bpa and TOAC at successive positions in the 3(10)-helix, radical-triplet interaction was observed in all cases. However, for the peptide where Bpa and TOAC are at positions 2 and 4 the rate of triplet quenching is lower than for the other peptides in the series. In addition, the radical-excited triplet complex in the quartet spin state was observed in a peptide containing fullerene (C(60)) as a triplet precursor and TOAC.