Phytyl Side Chain Research Articles

A relationship between amide hydrogen bond strength and quinone reduction potential: Implications for photosystem I and bacterial reaction center quinone function

A series of 11 simple phylloquinone derivatives, each lacking the extended phytyl side chain but featuring H-bond donor amides at one or both peri positions, were prepared and some salient physical properties were measured. A correlation between both IR frequency and NMR peak position, as indicators of internal H-bond strength, and the quinone half-wave reduction potential, was observed. These data are consistent with the prevailing hypothesis that quinone carbonyl H-bonding in general, and stronger H-bonds in particular, favorably bias the endogenous quinone’s electrochemical potential toward easier reduction.

Advantages and limitation of BODIPY as a probe for the evaluation of lipid peroxidation and its inhibition by antioxidants in plasma

Oxidative stress and the role of antioxidants are currently one of the most important subjects in the field of life science. In the present study, we assessed the oxidation of plasma lipids induced by free radicals and its inhibition by antioxidants with a fluorescence probe BODIPY. Vitamin E and C-depleted plasma was used to evaluate the inherent action of several antioxidants. BODIPY reacted with free radicals in plasma to emit fluorescence (ex. 510 nm, em. 520 nm), which was suppressed by the antioxidants in a concentration-dependent manner. However, the suppression of fluorescence emission by antioxidants did not always correlate quantitatively with the suppression of lipid peroxidation. For example, α-tocopherol suppressed BODIPY fluorescence but enhanced the peroxidation of plasma lipids in the absence of ascorbic acid. 2,2,5,7,8-Pentamethyl-6-chromanol, a vitamin E analogue without a phytyl side chain, almost completely suppressed both fluorescence emission and lipid peroxidation in the plasma. These results show that BODIPY can be used as a convenient probe for radical scavenging, but that care should be taken for the evaluation of antioxidant capacity.

α-Tocopheryl Succinate Induces Apoptosis in Prostate Cancer Cells in Part through Inhibition of Bcl-xL/Bcl-2 Function

Although the antitumor effect of alpha-tocopheryl succinate (vitamin E succinate) has been well demonstrated, its underlying mechanism remains elusive. This study provides evidence that inhibition of Bcl-xL/Bcl-2 function represents a major pathway whereby alpha-tocopheryl succinate mediates apoptosis induction in prostate cancer cells. In vitro data indicate that alpha-tocopheryl succinate was able to disrupt the binding of Bak BH3 peptide to Bcl-xL and Bcl-2 with IC50 of 26 microm, in line with its potency in antiproliferation. Treatment of PC-3 cells with this agent led to reduced association of Bcl-2 and Bcl-xL with Bak, leading to caspase-dependent apoptosis. Moreover, overexpression of Bcl-xL protected LNCaP cells from the apoptosis induction. This mechanistic finding provided a basis to develop potent Bcl-xL/Bcl-2 inhibitors. Docking of alpha-tocopheryl succinate into the Bak peptide-binding site indicates that it adopted a unique hairpin-shaped conformation for protein interactions. We rationalized that the hemisuccinate and the two proximal isopranyl units of the side chain played a crucial role in ligand anchoring and protein-ligand complex stabilization, respectively. However, exposure of the distal isopranyl unit to a polar environment might diminish the binding affinity of alpha-tocopheryl succinate. This premise was corroborated by a structure-activity analysis of a series of derivatives with truncated side chains and/or altered carboxyl terminus. This computer model predicted that the removal of the distal isopranyl unit from the side chain would improve binding affinity, leading to two agents with significantly higher potency in inhibiting Bak peptide binding and in suppressing prostate cancer cell proliferation.

Free radical oxidation (autoxidation) of alkenones and other microalgal lipids in seawater

The free radical oxidation (autoxidation) of long chain alkenones derived from haptophyte microalgae was studied in the laboratory in the presence of a radical initiator (di- tert-butyl nitroxide) and a radical enhancer ( tert-butyl hydroperoxide). Alkenones were autoxidized faster than analogues of other common marine lipids such as phytyl acetate, methyl oleate and cholesteryl acetate. Alkenone autoxidation rates increased in an approximately linear fashion with the number of double bonds present in the molecule and so autoxidation has the potential to affect values of U 37 K ′ . The process seems to involve allylic hydrogen abstraction and subsequent oxidation of the allylic radical to form products that can be analysed as alkene polyols after NaBH 4 reduction. Evidence for the autoxidation of lipids in seawater was obtained from analysis of organic matter in sediment traps deployed at the DYFAMED site in the northwestern Mediterranean Sea. High contents of specific autoxidation products of monounsaturated fatty acids and the chlorophyll phytyl side chain were recognized in samples collected during post-bloom conditions, but not during a phytoplankton bloom event. Biomarkers for autoxidation include the presence of 8- and 11-hydroxyoctadec- cis-9-enoic acids and various phytol diols and triols. These same samples also showed anomalously high U 37 K ′ values (equivalent to an inferred temperature change of at least 2 °C), consistent with the results of the autoxidation experiments, but opposite to that expected for any effect due to nutrient limitation on haptophyte cells. Our work demonstrates that autoxidation of lipids may occur in seawater and that this has the potential to affect alkenone distributions, especially where residence times of particles are long. In such circumstances this might lead to a warm bias in estimates of palaeotemperature derived from alkenone ratios in sediments.

Functional evidence for the involvement of Arabidopsis IspF homolog in the nonmevalonate pathway of plastid isoprenoid biosynthesis

There are two independent pathways, the cytosolic mevalonate (MVA) pathway and the plastid nonmevalonate (nonMVA) pathway, to synthesize isopentenyl diphosphate and dimethylallyl diphosphate in plants. Carotenoids and the phytyl side chain of chlorophylls are isoprenoids derived from the plastid nonMVA pathway. All enzymes involved in the nonMVA pathway have been identified in Escherichia coli. The E. coli IspF protein catalyzes a unique cyclization reaction to convert 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate into 2-C-methyl-D-erythritol 2,4-cyclodiphosphate in the nonMVA pathway. We have characterized an Arabidopsis T-DNA insertion mutant, ispF-1, which has a null mutation in the IspF gene. Homozygous ispF-1 mutants are albino lethal and the IspF transcripts are undetectable in these plants. Moreover, the ispF-1 mutant chloroplasts are filled with vesicles instead of thylakoids. Amino acid sequence alignment reveals that the IspF proteins are highly conserved between plants and bacteria. Interestingly, expression of the Arabidopsis IspF protein can rescue the lethal phenotype of an E. coli ispF mutant. These results indicate that the Arabidopsis IspF may share similar enzymatic mechanisms with the E. coli protein.

Determination of the urinary aglycone metabolites of vitamin K by HPLC with redox-mode electrochemical detection

We describe a method for the determination of the two major urinary metabolites of vitamin K as the methyl esters of their aglycone structures, 2-methyl-3-(3'-3'-carboxymethylpropyl)-1,4-naphthoquinone (5C-aglycone) and 2-methyl-3-(5'-carboxy-3'-methyl-2'-pentenyl)-1,4-naphthoquinone (7C-aglycone), by HPLC with electrochemical detection (ECD) in the redox mode. Urinary salts were removed by reversed-phase (C18) solid-phase extraction (SPE), and the predominantly conjugated vitamin K metabolites were hydrolyzed with methanolic HCl. The resulting carboxylic acid aglycones were quantitatively methylated with diazomethane and fractionated by normal-phase (silica) SPE. Final analysis was by reversed-phase (C18) HPLC with a methanol-aqueous mobile phase. Metabolites were detected by amperometric, oxidative ECD of their quinol forms, which were generated by postcolumn coulometric reduction at an upstream electrode. The assay gave excellent linearity (typically, r2 > or = 0.999) and high sensitivity with an on-column detection limit of < 3.5 fmol (< 1 pg). The interassay precision was typically 10%. Metabolite recovery was compared with that of an internal standard [2-methyl-3-(7'-carboxy-heptyl)-1,4-naphthoquinone] added to urine samples just before analysis. Using this methodology, we confirmed that the 5C- and 7C-aglycones were major catabolites of both phylloquinone (vitamin K1) and menaquinones (vitamin K2) in humans. We propose that the measurement of urinary vitamin K metabolite excretion is a candidate noninvasive marker of total vitamin K status.

Alpha and gamma tocopherol metabolism in healthy subjects and patients with end-stage renal disease

The metabolism of alpha and gamma tocopherol, the major components of vitamin E, have not been studied in uremic patients. The major pathway of tocopherol metabolism is via phytyl side chain oxidation, leaving carboxyethyl-hydroxychromans (CEHC) as metabolites. Alpha and gamma CEHC are water soluble, renally excreted, with known potent anti-inflammatory and antioxidative properties. We examined serum alpha and gamma tocopherol and respective CEHC concentrations in 15 healthy subjects and 15 chronic hemodialysis patients. Serum alpha tocopherol levels were similar in hemodialysis patients (12.03 +/- 1.34 microg/mL) and healthy subjects (11.21 +/- 0.20 microg/mL), while serum gamma tocopherol levels were significantly greater in hemodialysis patients (3.17 +/- 0.37 microg/mL) compared to healthy subjects (1.08 +/- 0.06 microg/mL, P < 0.0001). Serum alpha and gamma CEHC levels were tenfold and sixfold higher in hemodialysis patients compared to healthy subjects, respectively (both P < 0.0001). Serum alpha and gamma tocopherol levels and CEHC metabolites were also measured after supplementation of alpha- or gamma-enriched mixed tocopherols in both hemodialysis patients and healthy subjects. Tocopherol administration resulted in modest or nonsignificant changes in serum tocopherol concentrations, while markedly increasing serum CEHC concentrations in both healthy subjects and hemodialysis patients. Hemodialysis resulted in no change in the serum alpha or gamma tocopherol concentrations while decreasing serum alpha CEHC and gamma CEHC levels by 63% and 53%, respectively (both P = 0.001 versus predialysis). Fourteen-day administration of gamma-enriched but not alpha tocopherols lowered median C-reactive protein (CRP) significantly in hemodialysis patients (4.4 to 2.1 mg/L, P < 0.02). First, serum alpha and gamma CEHC accumulate in uremic patients compared to healthy subjects; second, supplementation with tocopherols dramatically increases serum CEHC levels in both healthy subjects and hemodialysis patients; and, finally, CEHC accumulation may mediate anti-inflammatory and antioxidative effects of tocopherols in hemodialysis patients.

Improved Synthesis of Vitamin K1.

AbstractFor Abstract see ChemInform Abstract in Full Text.

Biosynthesis of chloroplastidic and extrachloroplastidic terpenoids in liverwort cultured cells: 13C serine as a probe of terpene biosynthesis via mevalonate and non-mevalonate pathways.

Two terpenoid biosynthetic pathways, the mevalonate and non-mevalonate (glyceraldehyde phosphate-pyruvate) routes, were examined by feeding (13)C-labeled serines ([1-(13)C]- and [3-(13)C]-) to the cultured cells of the liverwort, Heteroscyphus planus. The labeling patterns observed in the isoprenoid unit of the biosynthetically (13)C-labeled stigmasterol corresponded to those expected from the mevalonate pathway, while those of the phytyl side chain corresponded to those from the non-mevalonate pathway. Thus, serine is a potential probe to determine the origin of terpenoid biosynthesis, in either the mevalonate or non-mevalonate pathway.

Improved Synthesis of Vitamin K1

With (E/Z)-isomeric phytyl halides as side-chain materials, vitamin K 1 is synthesized via a Diels–Alder reaction to activate the free bridgehead hydrogen of 3 for the alkylation and a retro-Diels–Alder reaction to eliminate cyclopentadiene from 2 in a high yield, in which the configuration of the double bond in the phytyl side-chain is retained.

Visible light-induced oxidation of lipid components of purple sulfur bacteria: a significant process in microbial mats

Visible light-induced degradation processes were studied in vitro in senescent cells of two purple sulfur bacteria ( Thiohalocapsa halophila and Halochromatium salexigens) isolated from microbial mats from Camargue (France). These reactions act on the phytyl side chain of bacteriochlorophyll- a and on palmitoleic and cis-vaccenic acids. The experiments also confirmed some previous results, i.e. the regiospecific enzymatic oxygenation of the allylic carbon 10 of cis-vaccenic acid in senescent cells of T. halophila. The subsequent detection of substantial amounts of photoproducts from the phytyl chain and mono-unsaturated fatty acids in the lipid extracts of the microbial mats from which these two strains were isolated clearly established the importance of photodegradation processes in this particular ecosystem. The presence of photoproducts of cis-vaccenic acid showed that under natural conditions photochemical reactions act extensively on senescent phototrophic bacteria. Significant amounts of 10-oxooctadecanoic and 10-hydroxy-11-oxooctadecanoic acids were also detected in the lipid extracts. The detection of these compounds, which result from the enzymatic oxidation of carbon 10 of cis-vaccenic acid, supports a strong T. halophila contribution to the microbial mats.

Cytochrome P450 ω-Hydroxylase Pathway of Tocopherol Catabolism: NOVEL MECHANISM OF REGULATION OF VITAMIN E STATUS

Postabsorptive elimination of the various forms of vitamin E appears to play a key role in regulation of tissue tocopherol concentrations, but mechanisms of tocopherol metabolism have not been elucidated. Here we describe a pathway involving cytochrome P450-mediated omega-hydroxylation of the tocopherol phytyl side chain followed by stepwise removal of two- or three-carbon moieties, ultimately yielding the 3'-carboxychromanol metabolite that is excreted in urine. All key intermediates of gamma-tocopherol metabolism via this pathway were identified in hepatocyte cultures using gas chromatography-mass spectrometry. NADPH-dependent synthesis of the initial gamma- and alpha-tocopherol 13'-hydroxy and -carboxy metabolites was demonstrated in rat and human liver microsomes. Functional analysis of several recombinant human liver P450 enzymes revealed that tocopherol-omega-hydroxylase activity was associated only with CYP4F2, which also catalyzes omega-hydroxylation of leukotriene B(4) and arachidonic acid. Tocopherol-omega-hydroxylase exhibited similar binding affinities but markedly higher catalytic activities for gamma-tocopherol than alpha-tocopherol, suggesting a role for this pathway in the preferential physiological retention of alpha-tocopherol and elimination of gamma-tocopherol. Sesamin potently inhibited tocopherol-omega-hydroxylase activity exhibited by CYP4F2 and rat or human liver microsomes. Since dietary sesamin also results in elevated tocopherol levels in vivo, this pathway appears to represent a functionally significant means of regulating vitamin E status.

Antioxidant Action of 2,2,4,6-Tetra-Substituted 2,3-Dihydro-5-hydroxybenzofuran Against Lipid Peroxidation: Effects of Substituents and Side Chain

With increasing evidence suggesting the involvement of oxidative stress in various disorders and diseases, the role of antioxidants in vivo has received much attention. 2,3-Dihydro-5-hydroxy-2,2-dipentyl-4,6-di- tert -butylbenzofuran (BO-653) was designed, synthesized and has been evaluated as a novel antiatherogenic drug. In order to further understand the action of BO-653 and also radical-scavenging antioxidants in general, the dynamics of inhibition of oxidation by BO-653 were compared with those of the related compounds, 2,3-dihydro-5-hydroxy-2,2-dimethyl-4,6-di- tert -butylbenzofuran (BOB), 2,3-dihydro-5-hydroxy-2,2,4,6-tetramethylbenzofuran (BOM), f -tocopherol and 2,2,5,7,8-pentamethyl-6-chromanol (PMC), aiming specifically at elucidating the effects of substituents and side chain length of the phenolic antioxidants. These five antioxidants exerted substantially the same reactivities toward radicals and antioxidant capacities against lipid peroxidation in organic solution. When compared with di-methyl side chains, the di-pentyl side chains of BO-653 reduced its inter-membrane mobility but exerted less significant effect than the phytyl side chain of f -tocopherol on the efficacy of radical scavenging within the membranes. Di- tert -butyl groups at both ortho-positions made BO-653 and BOB more lipophilic than di-methyl substituents and reduced markedly the reactivity toward Cu(II) and also the synergistic interaction with ascorbate. The results of the present study together with those of the previous work on the effect of substituents on the stabilities of aryloxyl radicals suggest that tert -butyl group is more favorable than methyl group as the substituent at the ortho-positions and that di-pentyl side chains may be superior to a phytyl side chain.

One-year seasonal survey of the chlorophyll photodegradation process in the northwestern Mediterranean Sea

Abstract Particulate samples from the water column were collected monthly from depths of 5–150 m, between May 1996 and March 1997, in the northwestern Mediterranean Sea (Ligurian Sea) as part of the DYFAMED project within the French JGOFS program. These samples were analyzed by gas chromatography-electron impact mass spectrometry for their phytol and 3-methylidene-3,7,11-trimethylhexadecan-1,2-diol (phytyldiol) content. The corresponding Chlorophyll Phytyl side chain Photodegradation Index, molar ratio of phytyldiol to phytol, was calculated and the mean amount of chlorophyll photodegraded within the euphotic zone estimated. Seasonal differences in the chlorophyll photodegradation process appear in the one-year study. The chlorophyll appeared more photodegraded in the surface water (generally more than 40% photodegraded at 5–10 m) than at the deep chlorophyll maximum (DCM) (40–50 m) observed in the summer stratified waters (about 20% photodegraded). This difference was attributed to the healthy state of the phytoplankton community (coincidence with the highest primary production levels) and to the lower intensity of irradiance at the DCM level. On the other hand, the bulk of the detrital chlorophyll (chlorophyll associated with phytodetritus, phaeopigments) undergoes photodegradation before it sinks out of the photic zone. However, in January (winter mixed water) the pigments exported towards the sea floor were less photodegraded. This is thought to result from a shorter period of residence of the pigments in the photic zone due to vertical convection and grazing activity of macrozooplankton (salps), which are producers of rapid sinking fecal pellets.

Tocopherols are metabolized in HepG2 cells by side chain ω-oxidation and consecutive β-oxidation

The metabolism of tocopherols by ω- and β-oxidation of the phytyl side chain has been inferred from the identification of the final products carboxyethyl-hydroxychromans (CEHC) and immediate precursors, α- and γ-carboxymethylbutyl-hydroxychromans (CMBHCs). This hypothesis is here corroborated by the identification of a further α-tocopherol metabolite, α-carboxymethylhexyl-hydroxychroman (α-CMHHC), and evidence for the involvement of a P 450-type cytochrome. HepG2 cells, when exposed to 100 μM all-rac-α-tocopherol, released α-CEHC, α-CMBHC, and α-CMHHC into the medium. The detection of those metabolites required pretreatment of the cells with α-tocopherol for 10 d. In contrast, analogous metabolites of γ and δ-tocopherol were detectable without any preconditioning, while corresponding metabolites of RRR-α-tocopherol could not be detected at all. The formation of α-CEHC from all-rac-α-tocopherol was enhanced up to 5-fold by pretreatment of the HepG2 cells with rifampicin, known to induce CYP3A-type cytochromes with the capability of catalyzing ω-oxidation. In contrast, clofibrate did not reveal any effect. This observation suggests that a CYP3A-type cytochrome initiates tocopherol metabolism by ω-oxidation. It further reveals that inducible ω-oxidation is the rate-limiting step in tocopherol metabolism. It is discussed that competition of microsomal ω-oxidation with specific binding by the α-tocopherol transfer protein (α-TTP) determines the metabolic fate of the individual tocopherols.

Labeling Patterns of Chloroplastidic Isoprenoids in Cultured Cells of Liverwort Ptychanthus striatus

Incorporation studies administering 2H- and 13C-labeled mevalonate (MVA) and 13C-labeled glucose to suspension cultured cells of the liverwort, Ptychanthus striatus, were carried out in order to examine the biosynthesis of the phytyl side-chain of chlorophyll a. Administration of 13C- and 2H-labeled MVA provided evidence for the involvement of the MVA pathway in the phytyl side-chain biosynthesis and preferential labeling of the farnesyl diphosphate (FPP)-derived portion. An alternate labeling pattern in the phytyl side-chain was observed which was slightly different to the non-equivalent labeling in other liverworts, such as Heteroscyphus planus and Lophocolea heterophylla and in the hornwort, Anthoceros punctatus. The labeling pattern observed after the administration of 13C-labeled glucose revealed the simultaneous involvement of the non-MVA pathway in the phytol biosynthesis of P. striatus cells.

Nonequivalent labeling of the phytyl side chain of chlorophyll a in callus of the hornwort Anthoceros punctatus.

Callus cultures of the hornwort Anthoceros punctatus were induced from the apical portions of the gametophytes. Calli can accumulate rosmarinic acid, which is suggested as an intermediate for anthocerotonic acid, a rare phenylpropanoid dimer with a cyclobutane ring, indicating that calli possess the ability to produce secondary metabolites found primarily in intact plants. Biosynthesis of chloroplastidic terpenoids of liverworts showed preferential labeling of the farnesyl diphosphate (FPP)-derived portion in the phytyl side chain of chlorophyll a (1) when calli of A. punctatus are incubated with (2)H- and (13)C-labeled mevalonate. This finding suggests either that cytoplasmic FPP (or isopentenyl diphosphate, IPP) is taken into chloroplasts and condensed with endogenous IPP derived from a nonmevalonate pathway, or that FPP is synthesized within chloroplasts from extraplastidically formed IPP (or mevalonate) and then condensed with endogenous IPP in a different subplastidic fraction.

The purification of chlorins from marine particles and sediments for nitrogen and carbon isotopic analysis

Methods are presented for the separation of chlorins from marine particles and sediments for nitrogen and carbon isotopic analysis. Carbon and nitrogen purities of 91 and 96%, respectively, were obtained for particulate chlorins, with recoveries of 88%. Sedimentary chlorins were obtained with carbon and nitrogen purities of 88 and 89%, respectively, and recoveries of 18%. The reproducibility of carbon and nitrogen isotopic analyses on particulate and sedimentary chlorins was 0.2‰. The low recovery of chlorins from sediments is primarily a result of their complex distribution, requiring that only the one or two most abundant chlorins from a sample are purified. The procedure for particulate samples includes ultrasonic extraction with solvents, two phase separations, and reversed- and normal-phase high-performance liquid chromatographic separations (HPLC). The procedure for sedimentary chlorins includes ultrasonic extraction by solvent, solid-phase extraction on silica gel, reversed-phase HPLC, size-exclusion chromatography, and normal-phase HPLC. The purification procedures require ca. 4 h for particulate samples, and ca. 8 h for sediment samples when two samples are processed simultaneously. The application of these methods to surficial Black Sea sediments demonstrated that different chlorins have distinct nitrogen and carbon isotopic ratios. The δ 15N differences are interpreted in terms of changes in the seasonal flux of material out of the euphotic zone, while the δ 13C differences are thought to derive from the presence or absence of the chlorin phytyl side-chain.

Can a high chlorophyllase activity bias the use of the phytyldiol versus phytol ratio (CPPI) for the monitoring of chlorophyll photooxidation in seawater?

The photodegradation rates of chlorophyll-a and phytol in both its esterified and free forms were followed in killed cells of Phaeodactylum tricornutum Bohlin, a diatom possessing a strong chlorophyllase activity. The Chlorophyll Phytyl side chain Photodegradation Index (CPPI), defined as the molar ratio of phytyldiol (a specific photoproduct of the phytyl chain) versus phytol, was used to estimate the chlorophyll-a photodegradation rate. Despite the strong chlorophyllase activity of this alga and the high photoreactivity of free phytol, the CPPI gave a good estimation of the chlorophyll-a photodegradation state. These estimates were good for the axenic and non-axenic incubations, since aerobic bacterial degradation processes act upon free phytyldiol and phytol without high selectivity.

ENDOR and special TRIPLE resonance spectroscopy of photoaccumulated semiquinone electron acceptors in the reaction centers of green sulfur bacteria and heliobacteria.

Photoaccumulation at 205 K in the presence of dithionite produces EPR signals in anaerobically prepared membranes from Chlorobium limicola and Heliobacterium chlorum that resemble the EPR spectrum of phyllosemiquinone (A1*-) photoaccumulated in photosystem I. We have used ENDOR and special TRIPLE resonance spectroscopy to demonstrate conclusively that these signals arise from menasemiquinone electron acceptors reduced by photoaccumulation. Hyperfine couplings to two protons H-bonded to the semiquinone oxygens have been identified by exchange of H. chlorum into D2O, and hyperfine couplings to the methyl group, and the methylene group of the phytyl side chain, of the semiquinone have also been assigned. The electronic structure of these menasemiquinones in these reaction centers is very similar to that of phyllosemiquinone in PSI, and shows a distorted electron spin density distribution relative to that of phyllosemiquinone in vitro. Special TRIPLE resonance spectrometry has been used to investigate the effect of detergents and oxygen on membranes of C. limicola. Triton X-100 and oxygen affect the menaquinone binding site, but n-dodecyl beta-D-maltoside preparations exhibit a relatively unaltered special TRIPLE spectrum for the photoaccumulated menasemiquinone.