Decrease In Light Absorption Research Articles

Dissipation of excess excitation energy of the needle leaves in Pinus trees during cold winters.

Photooxidative damage to the needle leaves of evergreen trees results from the absorption of excess excitation energy. Efficient dissipation of this energy is essential to prevent photodamage. In this study, we determined the fluorescence transients, absorption spectra, chlorophyll contents, chlorophyll a/b ratios, and relative membrane permeabilities of needle leaves of Pinus koraiensis, Pinus tabulaeformis, and Pinus armandi in both cold winter and summer. We observed a dramatic decrease in the maximum fluorescence (F m) and substantial absorption of light energy in winter leaves of all three species. The F m decline was not correlated with a decrease in light absorption or with changes in chlorophyll content and chlorophyll a/b ratio. The results suggested that the winter leaves dissipated a large amount of excess energy as heat. Because the cold winter leaves had lost normal physiological function, the heat dissipation depended solely on changes in the photosystem II supercomplex rather than the xanthophyll cycle. These findings imply that more attention should be paid to heat dissipation via changes in the photosystem complex structure during the growing season.

Spermine Selectively Inhibits High-Conductance but not Low-Conductance Mode of the Mitochondrial Permeability Transition Pore (MPTP)

Spermine is a biological organic polymer that has four primary amino groups. It is involved in the regulation of transcription, cell signalling and enzymatic activity. It has also been demonstrated that polyamines inhibit calcium-induced high amplitude mitochondrial swelling, suggesting their involvement in regulation of the mitochondrial Permeability Transition Pore (mPTP). Here we further investigated the mechanisms of polyamine inhibition of calcium-induced, cyclosporine A (CSA) sensitive mPTP.Experiments were performed using isolated rat liver mitochondria resuspended in sucrose based recording solution. mPTP was induced by the addition of calcium to energized mitochondria. mPTP activation was measured by three independent approaches: 1) calcium release from the mitochondria detected with calcium green; 2) mitochondrial membrane depolarization using TMRM probe and 3) mitochondrial swelling measured as a decrease of light absorbance. In control experiments, in the presence of the specific inhibitor CSA, mPTP inhibition was observed by all three approaches. However, in the presence of spermine (0.02 mM to 0.2 mM) only mitochondrial swelling was inhibited, while membrane depolarization and calcium release were not affected.These results suggest that spermine inhibited high-conductance mode of mPTP, which opening is required for induction of mitochondrial swelling in sucrose based media. However spermine did not inhibit low-conductance mode mPTP. Our data are consistent with possible distinct regulation and/or nature of the different modes of mPTP.

Effect of Pulsed Electric Fields Treatment and Mash Size on Extraction and Composition of Apple Juices

This study explored the effect of pulsed electric field (PEF) treatment (E=450 V/cm; tt=10 ms; E<3 kJ/kg) and apple mash size on juice yield, polyphenolic compounds, sugars, and malic acid. Juice yield increased significantly after PEF treatment of large mash (Y=71.4%) and remained higher than the juice yield obtained for a control small mash (45.6%). The acid sweet balance was not altered by PEF. A correlation was established between the decrease of light absorbance (control: 1.43; treated: 1.10) and the decline of native polyphenols yield due to PEF treatment (control: 9.6%; treated: 5.9% for small mash). An enhanced oxidation of phenolic compounds in cells due to electroporation of the inner cell membrane and the adsorption of the oxidized products on the mash may explain both the lower light absorbance and the lower native polyphenol concentration.

Improvement of efficiency droop of GaN-based light-emitting devices by a rear nitride reflector

GaN-based light-emitting devices (LEDs) with and without a rear distributed Bragg reflector (DBR) were grown by metal–organic chemical vapor deposition. The integrated electroluminescence (EL) intensity of LED with a rear nitride DBR showed a super-linear increase up to a current density of 135 A/cm 2 and the relative external quantum efficiency (EQE) kept monotonously increasing with increase in injection current density. For the LED without a rear DBR, however, the emission efficiency reached the maximum at a very low current density of 10 A/cm 2. The improvement is mainly attributed to reflection of light from the DBR to the “top side” and decrease in light absorption in the active region with increase in injection current density. Moreover, the improvement in relative EQE was attributed to the resonant cavity enhancing spontaneous emission at its resonant wavelength too.

Effects of Preheating of Powder Before Spark Plasma Sintering of Transparent MgAl2O4 Spinel

The effects of preheating of powder on microstructure and optical transmission for MgAl2O4 spinel ceramics fabricated by spark plasma‐sintering processing are investigated. The preheating of powder in air enhances light transmission, whereas it has no remarkable effects on the microstructure. A decrease in light absorption was also observed for the spinel sintered after the preheating, and carbon contamination during sintering was not observed.

Controlled release of PEG chain from gold nanorods: Targeted delivery to tumor

Gold nanorods exhibit strong absorbance of light in the near infrared region, which penetrates deeply into tissues. Since the absorbed light energy is converted into heat, gold nanorods are expected to act as a contrast agent for in vivo bioimaging and as a thermal converter for photothermal therapy. To construct a gold nanorod targeted delivery system for tumor a peptide substrate for urokinase-type plasminogen activator (uPA), expressed specifically on malignant tumors, was inserted between the PEG chain and the surface of the gold nanorods. In other words, we constructed PEG–peptide-modified gold nanorods. After mixing the gold nanorods with uPA, the PEG chain was released from the surface of the gold and subsequently nanorod aggregation took place. The formation of the aggregation was monitored as a decrease in light absorption at 900 nm. Tumor homogenate induced a significant decrease in this absorption. Larger amount of the PEG–peptide-modified gold nanorods bound to cells expressing uPA in vitro compared with control gold nanorods, which had scrambled sequence of the peptide. The PEG–peptide-modified gold nanorods showed higher accumulation in tumor than the control after they were injected intravenously into tumor-bearing mice, however, the density of the peptide on the surface of the gold nanorods was a key factor of their biodistributions. This targeted delivery system, which responds to uPA activity, is expected to be a powerful tool for tumor bioimaging and photothermal tumor therapy.

Photobleaching of camphorquinone during polymerization of dimethacrylate-based resins

Objective The aim of this study was to compare the photobleaching rate of CQ in different dental resins. Methods The photodecomposition rate of CQ/amine system in bis-GMA/TEGDMA, bis-EMA and UDMA polymerizing monomers was evaluated at different light intensities. The photobleaching of the CQ was studied by monitoring the decrease in light absorption as a function of continuous irradiation time. The absorption changes were assessed by recording the transmitted light that passed through samples of monomers containing CQ/amine. Results Complete photobleaching of CQ was observed in all the monomer tested and the rate constant for the photobleaching was proportional to the radiation intensity. Hydrogen abstraction from amines by the excited CQ state via electron transfer and direct hydrogen abstraction from monomer structures were involved in the CQ photoreduction. CQ was photobleached in the absence of coinitiator in a dimethacrylate monomer containing a carbamate functional group (UDMA). This behavior was attributed to the presence of labile hydrogen atoms in the UDMA monomer. The CQ photobleaching rate constant in UDMA containing CQ/amine was similar to that in UDMA in the absence of amine. Moreover, the efficiency of CQ to photoinitiate the polymerization of UDMA in the absence of amine demonstrated that the radicals derived from the UDMA monomer via hydrogen abstraction are highly reactive toward double bonds. Significance CQ photoinitiates the polymerization of the UDMA monomer in the absence of amine and the efficiency of this process is comparable to that of traditional bis-GMA and bis-EMA monomers activated with CQ/amine.

Photoinitiation rate profiles during polymerization of a dimethacrylate-based resin photoinitiated with camphorquinone/amine. Influence of initiator photobleaching rate

The photodecomposition of camphorquinone (CQ)/amine during the photo polymerization of a dimethacrylate-based resin under continuous irradiation was investigated in thick samples. The global CQ photoconsumption was measured by monitoring the decrease in light absorption as a function of irradiation time and the kinetics were satisfactory fitted to a first order expression where the rate constant of photobleaching was proportional to the irradiation intensity. In a thick sample, the photobleaching of the photoinitiator is accompanied by a deeper penetration of the light through the underlying layers. These gradients of photoinitiator concentration, light intensity and photoinitiation rate along the path of irradiation were calculated. The photodecomposition reaction was spatially inhomogeneous and the degree of nonuniformity increased with increased initial sample absorbance. The influence of the photobleaching process on the polymerization reaction was examined. The photobleaching rate of CQ was much slower than the polymerization rate and only 20% of the initial amount of CQ was consumed before the polymerization reaction had almost ceased. Results obtained in this research highlight the inherent interlinking of light attenuation and photobleaching rate in bulk polymerizing systems.

Preservation of photosynthetic electron transport from senescence-induced inactivation in primary leaves after decapitation and defoliation of bean plants

The comparative effects of decapitation and defoliation on the senescence-induced inactivation of photosynthetic activity in primary leaves of bean plants were investigated. Decapitation was performed during different phases of bean plant ontogenesis, immediately after the appearance of the 1st, 2nd, 3rd and 4th composite leaf. In addition, we examined a variant with primary leaves and stem with an apical bud, but without composite leaves, i.e. defoliated plants. Analyses of chlorophyll fluorescence, millisecond delayed fluorescence and absorption at 830nm in primary leaves were undertaken to investigate the alterations in photosystems II and I electron transport during the decapitation-induced delayed senescence in the non-detached leaves. Analysis of the OKJIP transients using the JIP-test (see [Strasser R, Srivastava A, Tsimilli-Michael M. Analysis of the chlorophyll a fluorescence transient. In: Papageorgiou G, Govindjee, editors. Chlorophyll a fluorescence: a signature of photosynthesis. The Netherlands: Kluwer Academic Publishers, 2004; pp. 321-362]) showed an increase in several biophysical parameters of photosystem II in decapitated plants, specifically, the density of active reaction centers on a chlorophyll basis, the yields of trapping and electron transport, and the performance index. We also observed a decrease in the absorbed light energy per reaction center. Such a decrease in light absorption could be a result of the photosystem II down regulation that appeared as an increase in Q(B)-non-reducing photosystem II centers. The effect was identical when all leaves except the primary leaves were removed. The variant with a preserved apical bud, the defoliated plant, showed values similar to those of decapitated plants with primary leaves only. The changes in the induction curves of the delayed fluorescence also indicated an acceleration of electron transport beyond photosystem II in the decapitated and in defoliated plants. In these plants, the photosystem I-driven electron transport was accelerated, and the size of the plastoquinone pool was enhanced. It was established that decapitation can retard the senescence of primary leaves, can expand leaf life span and can cause activation of both photosystems I and II electron transport. The decapitation procedure shows similarities to the process of defoliation. The overcompensation effect that is developed after defoliation could initially be manifested as an acceleration of the linear photosynthetic electron flow in the rest of the leaves.

Mitochondrial matrix metalloproteinase activation decreases myocyte contractility in hyperhomocysteinemia

Cardiomyocyte N-methyl-d-aspartate receptor-1 (NMDA-R1) activation induces mitochondrial dysfunction. Matrix metalloproteinase protease (MMP) induction is a negative regulator of mitochondrial function. Elevated levels of homocysteine [hyperhomocysteinemia (HHCY)] activate latent MMPs and causes myocardial contractile abnormalities. HHCY is associated with mitochondrial dysfunction. We tested the hypothesis that HHCY activates myocyte mitochondrial MMP (mtMMP), induces mitochondrial permeability transition (MPT), and causes contractile dysfunction by agonizing NMDA-R1. The C57BL/6J mice were administered homocystinemia (1.8 g/l) in drinking water to induce HHCY. NMDA-R1 expression was detected by Western blot and confocal microscopy. Localization of MMP-9 in the mitochondria was determined using confocal microscopy. Ultrastructural analysis of the isolated myocyte was determined by electron microscopy. Mitochondrial permeability was measured by a decrease in light absorbance at 540 nm using the spectrophotometer. The effect of MK-801 (NMDA-R1 inhibitor), GM-6001 (MMP inhibitor), and cyclosporine A (MPT inhibitor) on myocyte contractility and calcium transients was evaluated using the IonOptix video edge track detection system and fura 2-AM. Our results demonstrate that HHCY activated the mtMMP-9 and caused MPT by agonizing NMDA-R1. A significant decrease in percent cell shortening, maximal rate of contraction (-dL/dt), and maximal rate of relaxation (+dL/dt) was observed in HHCY. The decay of calcium transient amplitude was faster in the wild type compared with HHCY. Furthermore, the HHCY-induced decrease in percent cell shortening, -dL/dt, and +dL/dt was attenuated in the mice treated with MK-801, GM-6001, and cyclosporin A. We conclude that HHCY activates mtMMP-9 and induces MPT, leading to myocyte mechanical dysfunction by agonizing NMDA-R1.

Mitochondrial MMP activation decreases myocyte contractility in hyperhomocysteinemia.

Hyperhomocysteinemia (HHCY) is associated with mitochondrial dysfunction. Matrix metalloproteinase protease (MMP) activation causes mitochondrial dysfunction. We tested the hypothesis that HCY activates myocyte mitochondrial MMP (mtMMP), induces mitochondrial permeability transition (MPT), and causes contractile dysfunction. Mice were administered HCY (0.6g/L) in drinking water for 10 wks. The mtMMP activity was measured by zymography. MPT was measured by decrease in light absorbance at 540 nm. Cellular calcium transients were detected using IonOptix edge detection system. The effect of MK‐801(NMDA‐R1 blocker), GM6001 (MMP blocker), and cyclosporine A (MPT blocker) on myocyte contractility was evaluated. HHCY induced the mtMMP‐9 and caused MPT. Calcium decay was faster in WT compared to HHCY. A decrease in cell shortening, maximal rate of contraction (−dL/dt), and maximal rate of relaxation (+dL/dt) was observed in HHCY (Fig. 1). Furthermore, HHCY‐induced decrease in cell shortening, −dL/dt, +dL/dt was attenuated with MK‐801, GM6001, and cyclosporine A. We conclude that HHCY activates mtMMP‐9, increases MPT leading to decrease in contractility by activating NMDA‐R1. This study was supported by AHA post‐doctoral training to KSM and NIH to SCT.

Optical and electrical properties of terbium films as a function of hydrogen concentration

AbstractThe absorption of hydrogen in the rare earth metals produces interesting changes in their optical and electrical properties. In this work we study these properties on Tb films, 50 nm thick, as a function of hydrogen concentration. The Tb films are covered with a 15 nm thick Pd overcoat which allows hydrogenation while protecting the highly reactive Tb film from contamination. Electrical resistivity is measured using the van der Pauw method, the transmission spectra are measured in the visible region and the concentration using a quartz crystal microbalance. The maximum concentration reached was 2.95 H/Tb at a hydrogen pressure of 450 Torr. From the measured transmission spectra, using a Spectral Projected Gradient Method, we obtain the Tb film's complex refractive index. From this we calculate the real and imaginary parts of the dielectric function, ε1 and ε2, and follow their evolution as a function of hydrogen concentration. ε1 changes from negative values, characteristic of metals, to positive values characteristic of semiconductors and dielectrics. ε2 decreases indicating a significant decrease in light absorption. These results are similar to those observed on Dy, Gd, La and Y films. (© 2005 WILEY‐VCH Verlag GmbH &amp; Co. KGaA, Weinheim)

Effects of iron limitation on photosynthesis and carbohydrate metabolism in the Antarctic diatom Chaetoceros brevis (Bacillariophyceae)

Iron, one of the structural elements of organic components that play an essential role in photosynthesis and nitrogen assimilation of plants, is available at extremely low concentrations in large parts of the Southern Ocean's surface waters. We tested the hypothesis that photosynthesis is the primary target of iron stress in phytoplankton living in this specific environment, resulting in a reduced carbohydrate production. Cultures of a small Antarctic diatom, Chaetoceros brevis, were exposed to two different photon irradiances under iron-rich and iron-poor conditions. Under both light regimes growth rate was reduced only slightly by iron starvation, as expected because the iron requirement of a small-celled species such as C. brevis is low. Even so, iron-starved cells differed markedly from iron-replete cells: for low and high irradiance, respectively, they had a 20 and 27% lower content of light-harvesting pigments (chlorophyll a and c 2 and fucoxanthin), a 8 and 15% decrease in light absorption and a 15 and 17% decrease in quantum yield of photosystem II. The diurnal production of water-extractable carbohydrates was reduced by 28 and 31%, which resulted in a low supply of energy and carbon skeletons from these storage products. This may well have influenced protein synthesis. The nocturnal consumption of carbohydrates was also reduced, which, together with the almost proportional decrease in cellular C and N content, suggests that the C and N metabolism were tightly tuned in iron-stressed cultures. The decrease in C and N content correlated with a decrease in cell volume. Our results indicate that iron limitation is likely to affect the ability of phytoplankton to maintain high rates of protein synthesis within the deep wind-mixed layer of the Southern Ocean. In addition, growth at the surface could be inhibited by too much light: iron-poor cultures of C. brevis grown at low irradiance showed enhanced sensitivity to photoinhibition.

The Interaction of New Pyridylporphyrins with Bovine Serum Albumin

The interaction of meso-tetra(4-N-hydroxyethylpyridyl)porphyrin, meso-tetra(3-N-hydroxyethylpyridyl)porphyrin, and their zinc complexes with bovine serum albumin (BSA) was studied by electronic spectroscopy, CD, and equilibrium dialysis at pH 7.2. The titration of the porphyrins with BSA was accompanied by a decrease in light absorption and a bathochromic shift of the Soret band, as well as by the appearance of an isobestic point. The porphyrin interaction with BSA also led to the induction of positive CD spectra in the visible region, which is explained by the porphyrin sorption on the protein globule. The equilibrium dialysis helped in determining the stoichiometry of binding and the binding constants of the porphyrins under study with BSA using Scatchard plots. This interaction is nonspecific and reversible. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.

Absence of the Lhcb1 and Lhcb2 proteins of the light-harvesting complex of photosystem II - effects on photosynthesis, grana stacking and fitness.

We have constructed Arabidopsis thaliana plants that are virtually devoid of the major light-harvesting complex, LHC II. This was accomplished by introducing the Lhcb2.1 coding region in the antisense orientation into the genome by Agrobacterium-mediated transformation. Lhcb1 and Lhcb2 were absent, while Lhcb3, a protein present in LHC II associated with photosystem (PS) II, was retained. Plants had a pale green appearance and showed reduced chlorophyll content and an elevated chlorophyll a/b ratio. The content of PS II reaction centres was unchanged on a leaf area basis, but there was evidence for increases in the relative levels of other light harvesting proteins, notably CP26, associated with PS II, and Lhca4, associated with PS I. Electron microscopy showed the presence of grana. Photosynthetic rates at saturating irradiance were the same in wild-type and antisense plants, but there was a 10-15% reduction in quantum yield that reflected the decrease in light absorption by the leaf. The antisense plants were not able to perform state transitions, and their capacity for non-photochemical quenching was reduced. There was no difference in growth between wild-type and antisense plants under controlled climate conditions, but the antisense plants performed worse compared to the wild type in the field, with decreases in seed production of up to 70%.

Photoacoustic and filter‐based ambient aerosol light absorption measurements: Instrument comparisons and the role of relative humidity

Ambient measurements are reported of aerosol light absorption from photoacoustic and filter‐based instruments (aethalometer and a particle soot absorption photometer (PSAP)) to provide insight on the measurement science. Measurements were obtained during the Big Bend Regional Aerosol and Visibility Observational Study at the Big Bend National Park in South Texas. The aethalometer measurements of black carbon concentration at this site correlate reasonably well with photoacoustic measurements of aerosol light absorption, with a slope of 8.1 m2/g and a small offset. Light absorption at this site never exceeded 2.1 Mm−1 during the month of collocated measurements. Measurements were also obtained, as a function of controlled relative humidity between 40% and 90%, during the Photoacoustic IOP in 2000 at the Department of Energy Southern Great Plains Cloud and Radiation Testbed site (SGP). PSAP measurements of aerosol light absorption correlated very well with photoacoustic measurements, but the slope of the correlation indicated the PSAP values were larger by a factor of 1.61. The photoacoustic measurements of light absorption exhibited a systematic decrease when the RH increased beyond 70%. This apparent decrease in light absorption with RH may be due to the contribution of mass transfer to the photoacoustic signal. Model results for the limiting case of full water saturation are used to evaluate this hypothesis. A second PSAP measured the light absorption for the same humidified samples, and indicated very erratic response as the RH changed, suggesting caution when interpreting PSAP data under conditions of rapid relative humidity change.

Angular and spectral sensitivity of fly photoreceptors. I. Integrated facet lens and rhabdomere optics

Three optical components of a fly's eye determine the angular sensitivity of the photoreceptors: the light diffracting facet lens, the wave-guiding rhabdomere and the light-absorbing visual pigment in the rhabdomere. How the integrated optical system of the fly eye shapes the angular sensitivity curves is quantitatively analyzed in five steps: (1) scalar diffraction theory for low Fresnel-number lenses is applied to four different facet lenses, with diameter 10, 20, 40, and 80 micro m, respectively, assuming a constant F-number of 2.2; (2) optical waveguide theory is used to calculate waveguide modes propagating in circular cylindrical rhabdomeres with diameter 1.0, 2.0, and 4.0 micro m, respectively; (3) the excitation of waveguide modes is studied with the tip of the waveguide positioned in the focal plane as well as outside this plane; (4) the light absorption from the various propagated modes by the visual pigment in the rhabdomere is calculated as a function of the angle of the incident light wave; and (5) the angular sensitivity of the photoreceptor is obtained by normalizing the total light absorption. Four wavelengths are considered: 300, 400, 500 and 600 nm. The analysis shows that the wavelength dependency of the lens diffraction is strongly compensated by that of the waveguide modes, an effect which is further enhanced by the decrease in light absorption when the mode number increases. The angular sensitivity of fly photoreceptors is robust to defocus and largely wavelength independent for all except very slender rhabdomeres.

Screening Assays for the Mitochondrial Permeability Transition Using a Fluorescence Multiwell Plate Reader

Opening of permeability transition (PT) pores in the mitochondrial inner membrane causes the mitochondrial permeability transition (MPT) and leads to mitochondrial swelling, membrane depolarization, and release of intramitochondrial solutes. Here, our aim was to develop high-throughput assays using a fluorescence plate reader to screen potential inducers and blockers of the MPT. Isolated rat liver mitochondria (0.5 mg/ml) were incubated in multiwell plates with tetramethylrhodamine methyl ester (TMRM, 1 μM), a potential-indicating fluorophore, and Fluo-5N (1 μM), a low-affinity Ca2+ indicator. Incubation led to mitochondrial polarization, as indicated by uncoupler-sensitive quenching of the red TMRM fluorescence. CaCl2 (100 μM) addition led to ruthenium red-sensitive mitochondrial Ca2+ uptake, as indicated by green Fluo-5N fluorescence. After Ca2+ accumulation, mitochondria depolarized, released Ca2+ into the medium, and began to swell. This swelling was monitored as a decrease in light absorbance at 620 nm. Swelling, depolarization, and Ca2+ release were prevented by cyclosporin A (1 μM), confirming that these events represented the MPT. Measurements of Ca2+, mitochondrial membrane potential, and swelling could be made independently from the same wells without cross interference, and all three signals could be read from every well of a 48-well plate in about 1 min. In other experiments, mitochondria were ester-loaded with carboxydichlorofluorescein (carboxy-DCF) during the isolation procedure. Release of carboxy-DCF after PT pore opening led to an unquenching of green carboxy-DCF fluorescence occurring simultaneously with swelling. By combining measurements of carboxy-DCF release, Ca2+ uptake, membrane potential, and swelling, MPT inducers and blockers can be distinguished from uncouplers, respiratory inhibitors, and blockers of Ca2+ uptake. This high-throughput multiwell assay is amenable for screening panels of compounds for their ability to promote or block the MPT.

Comparison of IgG preparations by a turbidimetric assay of opsonizing capacity

A convenient turbidimetric phagocytosis assay was applied for the functional comparison of various intravenous IgG preparations. Staphylococcus aureus (Oxford) was opsonized by the immunoglobulin samples in the presence of an IgG deficient serum as a source of complement. The opsonized bacteria were subjected to phagocytosis by neutrophil granulocytes isolated from healthy adults. The time course of phagocytosis was monitored by the decrease of light absorbance at 400 nm. Changes in light absorbance during a 15 min period of opsonophagocytosis (Δ E 400) were expressed as a percentage of Δ E 400 obtained by a reference IgG preparation. The opsonizing effect of five commercially available i.v. IgG preparations was compared. Three different preparations containing whole, non-modified IgG molecules had a comparable opsonizing effect while a further one prepared by propiolacton modification displayed a reduced activity (52%) of the reference preparation, taken as 100%. A preparation consisting of IgG molecules without an Fc-region proved to be practically ineffective (8.7%).

A Protein Radical and Ferryl Intermediates Are Generated by Linoleate Diol Synthase, a Ferric Hemeprotein with Dioxygenase and Hydroperoxide Isomerase Activities

Linoleate diol synthase (LDS) was isolated as a hemeprotein from the fungus Gaeumannomyces graminis. LDS converts linoleate sequentially to 8R-hydroperoxylinoleate (8-HPODE) through an 8-dioxygenase by insertion of molecular oxygen and to 7S,8S-dihydroxylinoleate through a hydroperoxide isomerase by intramolecular oxygen transfer. Light absorption and EPR spectra of LDS indicated that the heme iron was ferric and mainly high spin. Oxygen consumption during catalysis started after a short time lag which was reduced by 8-HPODE. Catalysis declined due to suicide inactivation. Stopped flow studies with LDS and 8-HPODE at 13 degreesC showed a rapid decrease in light absorption at 406 nm within 35 ms with a first order rate constant of 90-120 s-1. Light absorption at 406 nm then increased at a rate of approximately 4 s-1, whereas the absorption at 421 nm increased after a lag time of approximately 5 ms at a rate of approximately 70 s-1. EPR spectra at 77 K of LDS both with linoleic acid and 8-HPODE showed a transient doublet when quenched after incubation on ice for 3 s (major hyperfine splitting 2.3 millitesla; g = 2.005), indicating a protein radical. The relaxation properties of the protein radical suggested interaction with a metal center. 8-HPODE generated about twice as much radical as linoleic acid, and the 8-HPODE-induced radical appeared to be stable. Our results suggest that LDS may form, in analogy with prostaglandin H synthases, ferryl intermediates and a protein radical during catalysis.