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.     

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.     

The inter-relationship between triplet energies and spin chemistry.

Triplet energies play a considerable role in optical spectroscopy, and can be determined from phosphorescence or the quenching thereof. Their role in spin chemistry may not be as obvious, but the triplet state has always had an important function or utility, namely of reaction intermediates such as radical pairs, their precursors, of carbenes, and of the final products. In situ NMR spectroscopy represents a useful tool to explore certain properties of the triplet state, especially in cases with no phosphorescence. The 'phase' of CIDNP resonances, i.e., emission or enhanced absorption, reflects the spin selectivity of electron transfer reactions. In radical ion pairs the spin selectivity is determined by the relation between the change of the standard free enthalpy DeltaG degrees during the electron back transfer and the triplet energies (E(T)) of the products. If triplet recombination is energetically feasible (DeltaG degrees > E(T)), it is typically the more efficient process in agreement with the Marcus theory.     

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.     

Whole cell nuclear magnetic resonance characterization of two photochemically active states of the photosynthetic reaction center in heliobacteria

Applying photo-CIDNP (photochemically induced dynamic nuclear polarization) MAS (magic-angle spinning) nuclear magnetic resonance to whole cells of Heliobacillus (Hb.) mobilis, we demonstrate that heliobacterial reaction centers are operational in two different states as indicated by the occurrence of a light-induced spin-correlated radical pair. A culture maintained anaerobically is called "Braunstoff" (German for "brown substance"). After exposure to oxygen, Braunstoff is converted to "Grünstoff" ("green substance") as indicated by a color change due to the conversion of BChl g to Chl a(F). It is shown that electron transfer occurs symmetrically via both branches of cofactors in both forms. The donor and acceptor cofactors remain identical and unchanged upon conversion, while the intermediate accessory cofactors are transformed from BChl g to Chl a(F). The donor triplet state in Braunstoff is localized on the special pair donor and lives for 100 μs, demonstrating the absence of nearby carotenoids. In Grünstoff, the donor triplet becomes mobile and appears to be formed on an accessory cofactor.     

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.     

Flash photolysis of misonidazole and metronidazole

Laser flash photolysis at 355 nm of misonidazole or metronidazole in aqueous solutions produced the relatively long-lived nitro radical anion as the only observable transient species. When 266 nm excitation was used, a small yield of solvated electron was observed. It is suggested that the nitroimidazole first undergoes photoionization and the photoelectrons are scavenged by ground state nitroimidazole molecules to produce the nitro radical anion. Alternatively, added EDTA or carbonate ion acted as an electron donor to the excited state nitroimidazole molecule, thereby increasing the yield of nitro radical anion. The transient yield from metronidazole was about half that from misonidazole, while the phosphorescence intensity of metronidazole in an ethanol glass was about 20 times that of misonidazole. The misonidazole n, pi* triplet state is more easily reduced than that of metronidazole and, in the presence of an electron donor, the radical anion is postulated to result from electron transfer to the triplet state of the nitroimidazole.     

Light-activated cryptochrome reacts with molecular oxygen to form a flavin-superoxide radical pair consistent with magnetoreception

Cryptochromes are flavin-based photoreceptors occurring throughout the biological kingdom, which regulate growth and development in plants and are involved in the entrainment of circadian rhythms of both plants and animals. A number of recent theoretical works suggest that cryptochromes might also be the receptors responsible for the sensing of the magnetic field of the earth (e.g. in insects, migratory birds, or migratory fish). Cryptochromes undergo forward light-induced reactions involving electron transfer to excited state flavin to generate radical intermediates, which correlate with biological activity. Here, we give evidence of a mechanism for the reverse reaction, namely dark reoxidation of protein-bound flavin in Arabidopsis thaliana cryptochrome (AtCRY1) by molecular oxygen that involves formation of a spin-correlated FADH^•–superoxide radical pair. Formation of analogous radical pairs in animal cryptochromes might enable them to function as magnetoreceptors.     

Calibration of a resonance energy transfer imaging system.

A quantitative technique for the nondestructive visualization of nanometer scale intermolecular separations in a living system is described. A calibration procedure for the acquisition and analysis of resonance energy transfer (RET) image data is outlined. The factors limiting RET imaging of biological samples are discussed. Measurements required for the calibration include: (a) the spectral sensitivity of the image intensifier (or camera); (b) the transmission spectra of the emission filters; and (c) the quantum distribution functions of the energy transfer pair measured in situ. Resonance energy transfer imaging is demonstrated for two DNA specific dyes. The Förster critical distance for energy transfer between Hoechst 33342 (HO) and acridine orange (AO) is 4.5 +/- 0.7 nm. This distance is slightly greater than the distance of a single turn of the DNA helix (3.5 nm or approximately 10 base pairs), and is well below the optical diffraction limit. Timed sequences of intracellular energy transfer reveal nuclear structure, strikingly similar to that observed with confocal and electron microscopy, and may show the spatial distribution of eu- and hetero- chromatin in the interphase nuclei.     

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.     

Fluorescence resonance energy transfer study of the associative state of membrane-bound complexes of complement proteins C5b-8

Human complement protein C8 was labeled with the fluorescent chromophores fluorescein-5-isothiocyanate (FITC), 3-(4-isothiocyanatophenyl)-7-diethylamine-4-methyl coumarin (IPM), eosin-5-isothiocyanate (EOS), or Texas Red (sulforhodamine-101-sulfonyl chloride; TR) with only minor reduction in the specific hemolytic activity of the protein. The distribution of C5b-8 complexes bound to sheep erythrocyte membranes was investigated by monitoring fluorescence resonance energy transfer (RET) between the following RET donor/acceptor pairs of labeled C8: FITC-C8/EOS-C8, IPM-C8/EOS-C8, and FITC-C8/TR-C8. On binding to membranes containing pre-formed C5b67 complexes, specific RET was detected for each of the donor/acceptor pairs of labeled C8 investigated. In contrast, no energy transfer was observed for these RET donor/acceptor pairs of labeled C8 incubated in the presence of control membranes or in membrane-free solution. On the basis of a consideration of the transfer efficiency that would be expected for donor/acceptor pairs of labeled C8 that were uniformly dispersed on the membrane surface, these results suggest that C5b-8 complexes are aggregated into polymeric clusters when membrane-bound. The efficiency of donor-C8 to acceptor-C8 RET--and the hemolytic activity of membrane-bound C5b-8 (in the absence of C9)--are both related to the surface density of membrane-bound C5b67, suggesting that the physical clustering of the membrane-inserted C5b-8 complex may be related to the expression of its cytolytic activity.     

Radical reactions of thiamin pyrophosphate in 2-oxoacid oxidoreductases

Thiamin pyrophosphate (TPP) is essential in carbohydrate metabolism in all forms of life. TPP-dependent decarboxylation reactions of 2-oxo-acid substrates result in enamine adducts between the thiazolium moiety of the coenzyme and decarboxylated substrate. These central enamine intermediates experience different fates from protonation in pyruvate decarboxylase to oxidation by the 2-oxoacid dehydrogenase complexes, the pyruvate oxidases, and 2-oxoacid oxidoreductases. Virtually all of the TPP-dependent enzymes, including pyruvate decarboxylase, can be assayed by 1-electron redox reactions linked to ferricyanide. Oxidation of the enamines is thought to occur via a 2-electron process in the 2-oxoacid dehydrogenase complexes, wherein acyl group transfer is associated with reduction of the disulfide of the lipoamide moiety. However, discrete 1-electron steps occur in the oxidoreductases, where one or more [4Fe-4S] clusters mediate the electron transfer reactions to external electron acceptors. These radical intermediates can be detected in the absence of the acyl-group acceptor, coenzyme A (CoASH). The π-electron system of the thiazolium ring stabilizes the radical. The extensively delocalized character of the radical is evidenced by quantitative analysis of nuclear hyperfine splitting tensors as detected by electron paramagnetic resonance (EPR) spectroscopy and by electronic structure calculations. The second electron transfer step is markedly accelerated by the presence of CoASH. While details of the second electron transfer step and its facilitation by CoASH remain elusive, expected redox properties of potential intermediates limit possible scenarios. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Kinetic model of the effect of dehydration on electron transfer from membrane-bound cytochrome c to the photosynthetic reaction center

Amplitude characteristics and kinetics of laser-induced oxidation of high-potential cytochrome CH by a photosynthetic reaction center (RC) were investigated in Ectothiorhodospira shaposhnikovii chromatophore preparations of various humidity. It is shown that the diminuition of the amount of oxidized cytochrome and the decrease of the rate of the reaction on lowering the preparation humidity can be explained in terms of the concept of conformation-controlled electron transfer within the CH-RC complex. A model is suggested which predicts that the reversible transition of the complex from one conformational state which allows electron transfer ("contact" state) to the other in which the transfer is impossible ("non-contact" state) is the result of drying (or low temperature) induced changes in the electron tunnelling path in the region of "contact" of the cytochrome CH and RC protein globules.     

Mechanistic studies of the radical SAM enzyme spore photoproduct lyase (SPL)

Spore photoproduct lyase (SPL) repairs a special thymine dimer 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct or SP at the bacterial early germination phase. SP is the exclusive DNA photo-damage product in bacterial endospores; its generation and swift repair by SPL are responsible for the spores' extremely high UV resistance. The early in vivo studies suggested that SPL utilizes a direct reversal strategy to repair the SP in the absence of light. The research in the past decade further established SPL as a radical SAM enzyme, which utilizes a tri-cysteine CXXXCXXC motif to harbor a [4Fe-4S] cluster. At the 1+ oxidation state, the cluster provides an electron to the S-adenosylmethionine (SAM), which binds to the cluster in a bidentate manner as the fourth and fifth ligands, to reductively cleave the CS bond associated with the sulfonium ion in SAM, generating a reactive 5'-deoxyadenosyl (5'-dA) radical. This 5'-dA radical abstracts the proR hydrogen atom from the C6 carbon of SP to initiate the repair process; the resulting SP radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired TpT. SAM is suggested to be regenerated at the end of each catalytic cycle; and only a catalytic amount of SAM is needed in the SPL reaction. The H atom source for the back donation step is suggested to be a cysteine residue (C141 in Bacillus subtilis SPL), and the H-atom transfer reaction leaves a thiyl radical behind on the protein. This thiyl radical thus must participate in the SAM regeneration process; however how the thiyl radical abstracts an H atom from the 5'-dA to regenerate SAM is unknown. This paper reviews and discusses the history and the latest progress in the mechanistic elucidation of SPL. Despite some recent breakthroughs, more questions are raised in the mechanistic understanding of this intriguing DNA repair enzyme. This article is part of a Special Issue entitled: Radical SAM enzymes and Radical Enzymology.     

Low-temperature pulsed EPR study at 34 GHz of the triplet states of the primary electron Donor P865 and the carotenoid in native and mutant bacterial reaction centers of Rhodobacter sphaeroides

The photosynthetic charge separation in bacterial reaction centers occurs predominantly along one of two nearly symmetric branches of cofactors. Low-temperature EPR spectra of the triplet states of the chlorophyll and carotenoid pigments in the reaction center of Rhodobacter sphaeroides R-26.1, 2.4.1 and two double-mutants GD(M203)/AW(M260) and LH(M214)/AW(M260) have been recorded at 34 GHz to investigate the relative activities of the "A" and "B" branches. The triplet states are found to derive from radical pair and intersystem crossing mechanisms, and the rates of formation are anisotropic. The former mechanism is operative for Rb. sphaeroides R-26.1, 2.4.1, and mutant GD(M203)/AW(M260) and indicates that A-branch charge separation proceeds at temperatures down to 10 K. The latter mechanism, derived from the spin polarization and operative for mutant LH(M214)/AW(M260), indicates that no long-lived radical pairs are formed upon direct excitation of the primary donor and that virtually no charge separation at the B-branch occurs at low temperatures. When the temperature is raised above 30 K, B-branch charge separation is observed, which is at most 1% of A-branch charge separation. B-branch radical pair formation can be induced at 10 K with low yield by direct excitation of the bacteriopheophytin of the B-branch at 590 nm. The formation of a carotenoid triplet state is observed. The rate of formation depends on the orientation of the reaction center in the magnetic field and is caused by a magnetic field dependence of the oscillation frequency by which the singlet and triplet radical pair precursor states interchange. Combination of these findings with literature data provides strong evidence that the thermally activated transfer step on the B-branch occurs between the primary donor, P865, and the accessory bacteriochlorophyll, whereas this step is barrierless down to 10 K along the A-branch.     

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.     

Photoinduced electron transfer in singly labeled thiouredopyrenetrisulfonate azurin derivatives.

A novel method for the initiation of intramolecular electron transfer reactions in azurin is reported. The method is based on laser photoexcitation of covalently attached thiouredopyrenetrisulfonate (TUPS), the reaction that generates the low potential triplet state of the dye with high quantum efficiency. TUPS derivatives of azurin, singly labeled at specific lysine residues, were prepared and purified to homogeneity by ion exchange HPLC. Transient absorption spectroscopy was used to directly monitor the rates of the electron transfer reaction from the photoexcited triplet state of TUPS to Cu(II) and the back reaction from Cu(I) to the oxidized dye. For all singly labeled derivatives, the rate constants of copper ion reduction were one or two orders of magnitude larger than for its reoxidation, consistent with the larger thermodynamic driving force for the former process. Using 3-D coordinates of the crystal structure of Pseudomonas aeruginosa azurin and molecular structure calculation of the TUPS modified proteins, electron transfer pathways were calculated. Analysis of the results revealed a good correlation between separation distance from donor to Cu ligating atom (His-N or Cys-S) and the observed rate constants of Cu(II) reduction.     

Inter- and intra-molecular electron transfer in the cytochrome bc(1) complex

In this review, we compare the intra-molecular and inter-molecular electron transfer rate constants of the high-potential branch of the cytochrome bc(1) complex. Several methods such as the conventional stopped-flow spectroscopy, pH-induced electron transfer, photoactivated ruthenium complex induced electron transfer and photoreleaseable caged quinol, have been used to determine reaction rates between redox centers in an attempt to elucidate the reaction mechanism of this vital energy conserving complex. Since the most active pure cytochrome bc(1) complex has a turnover number of 800 s(-1), any step with a rate constant much larger than this will not be rate-limiting. The most likely rate-limiting step is the cytochrome b redox state governed movement of the head domain of iron-sulfur protein from its electron-accepting site ("fixed" or "b-state" position) to its electron donating site ("c(1)-state" position).