A backflow of electrons around photosystem II in Chlorella cells.

A study was made with a modulated oxygen electrode of the effect of variations of oxygen concentration on photosynthetic oxygen evolution from algal cells. When Chlorella vulgaris is examined with a modulated 650 nm light at 22 degrees C, both the oxygen yield and the phase lag between the modulated oxygen signal and the light modulations have virtually constant values between 800 and 120 ergs . cm-1 . s-1 if the bathing medium is in equilibrium with the air. Similar results are obtained at 32 degrees C between 1600 and 120 ergs . cm-2 . s-1. Under anaerobic conditions both the oxygen yield and the phase lag decrease if the light intensity is lowered below about 500 ergs . cm-2 . s-1 at 22 degrees C or about 1000 ergs . cm-2 . s-1 at 32 degrees C. A modulated 706 nm beam also gives rise to these phenomena but only at significantly lower rates of oxygen evolution. The cells of Anacystis nidulans and Porphyridium cruentum appear to react in the same way to anaerobic conditions as C. vulgaris. An examination of possible mechanisms to explain these results was performed using a computer simulation of photosynthetic electron transport. The simulation suggests that a backflow of electrons from a redox pool between the Photosystems to the rate-limiting reaction between Photosystem II and the water-splitting act can cause a decrease in oxygen yield and phase lag. If the pool between the Photosystems is in a very reduced state a significant cyclic flow is expected, whereas if the pool is largely oxidized little or no cyclic flow should occur. It is shown that the effects of 706 nm illumination and removal of oxygen can be interpreted in accordance with these proposals. Since a partial inhibition of oxygen evolution by 3-(3.4-dichlorophenyl)-1,1-dimethylurea (10(-8) M) magnifies the decreases in oxygen yield and phase lag, it is proposed that the pool which cycles back electrons is in front of the site of 3-(3,4-dichlorophenyl)-1,1-dimethylurea inhibition and is probably the initial electron acceptor pool after Photosystem II.     

Resonance Raman study on photoreduction of cytochrome c oxidase: distinction of cytochromes a and a3 in the intermediate oxidation states

Occurrence of photoreduction of bovine cytochrome c oxidase was confirmed with the difference absorption spectra and oxygen consumption measurements for the enzyme irradiated with laser light at 406.7, 441.6, and 590 nm. The resonance Raman spectra were obtained under the same experimental conditions as those adopted for the measurements of oxygen consumption and difference absorption spectra. The photoreduction was more effective upon irradiation at shorter wavelengths and was irreversible under anaerobic conditions. However, upon aeration into the cell, the original oxidized form was restored. It was found that aerobic laser irradiation produces a photo steady state of the catalytic dioxygen reduction and that the Raman scattering from this photo steady state probes cytochrome a2+ and cytochrome a3(3)+ separately upon excitations at 441.6 and 406.7 nm, respectively. The enzyme was apparently protected from the photoreduction in the spinning cell with the spinning speed between 1 and 1500 rpm. These results were explained satisfactorily with the reported rate constant for the electron transfer from cytochrome a to cytochrome a3 (0.58 s-1) and a comparable photoreduction rate of cytochrome a. The anaerobic photoreduction did give Raman lines at 1666 and 214 cm-1, which are characteristic of the ferrous high-spin cytochrome a3(2)+, but they were absent under aerobic photoreduction. The formyl CH = O stretching mode of the a3 heme was observed at 1671 cm-1 for a2+a3(2)+CO but at 1664 cm-1 for a2+a3(2)+CN-, indicating that the CH = O stretching frequency reflects the pi back-donation to the axial ligand similar to the oxidation state marker line (v4).     

Photodamage to Pigment in the Photosystem Ⅱ Reaction Center D1/D2/Cytochrome b559 Complex

Strong light (800 μmol photons/m2 per s)-induced bleaching of the pigment in the isolated photosystem Ⅱ reaction center (PSII RC) under aerobic conditions (in the absence of electron donors or acceptors) was studied using high-pressure liquid chromatography (HPLC), absorption spectra, 77K fluorescence spectra and resonance Raman spectra. Changes in pigment composition of the PSll RC as determined by HPLC after light treatment were as follows: with increasing illumination time chlorophyll (Chi) a and β-carotene (β-car)content decreased. However, decreases in pheophytin (Pheo) could not be observed because of the mixture of the Pheo formed by degraded chlorophyll possibly. On the basis of absorption spectra, it was determined that, with a short time of illumination, the initial bleaching occurred maximally at 680 nm but that with increasing illumination time there was a blue shift to 678 nm. It was suggested that P680 was destroyed initially, followed by the accessory chlorophyll. The activity of P680 was almost lost after 10 min light treatment. Moreover, the bleaching of Pheo and β-car was observed at the beginning of illumination.After illumination, the fluorescence emission intensity changed and the fluorescence maximum blue shifted,showing that energy transfer was disturbed. Resonance Raman spectra of the PSII RC excited at 488.0 and 514.5 nm showed four main bands, peaking at 1 527 cm-1 (υ1), 1 159 cm-1 (υ2), 1 006 cm-1 (υ3), 966 cm-1 (υ4) for 488.0 nm excitation and 1 525 cm-1 (υ1), 1 159 cm-1 (υ2), 1 007 cm-1 (υ3), 968 cm-1 (υ4) for 514.5 nm excitation.It was confirmed that two spectroscopically different β-car molecules exist in the PSII RC. After light treatment for 20 min, band positions and bandwidths were unchanged. This indicates that carotenoid configuration is not the parameter that regulates photoprotection in the PSII RC.     

Resonance Raman spectroscopy of pyridoxal Schiff bases

Resonance Raman (RR) spectra are reported for amino acid and amine adducts of pyridoxal 5'-phosphate (PLP) and 5'-deoxypyridoxal (5'-dPL) in aqueous solution. For the valine adducts, a detailed study has been carried out on solutions at pH and pD 5, 9, and 13, values at which the pyridine and imine protons are successively ionized, and on the adducts formed from 15N-valine, alpha-deuterovaline, and N-methyl-PLP. Good quality spectra were obtained, despite the strong fluorescence of pyridoxal Schiff bases, by adding KI as a quencher, and by exciting the molecules on the blue side of their absorption bands: 406.7 nm (cw Kr+ laser) for the pH 5 and 9 species (lambda max = 409 and 414 nm), and 354.7 nm (pulsed YAG laser, third harmonic) for the pH 13 species (lambda max = 360 nm). A prominent band at 1646 cm-1 is assigned to the imine C=N stretch via its 13 cm-1 15N shift. A 12 cm-1 down-shift of the band in D2O confirms that the Schiff base linkage is protonated at pH 9. Deprotonation at pH 13 shifts VC = N from 1646 to 1629 cm-1, values typical of conjugated Schiff bases. The strongest band in the spectrum, at 1338 cm-1, shifts to 1347 cm-1 upon pyridine protonation at pH 5, and is assigned to a ring mode with a large component of phenolate C-O stretch. A shoulder on its low-frequency side is assigned to the C4-C4' stretch. Large enhancements of these modes can be understood qualitatively in terms of the dominant resonance structures contributing to the ground and resonant excited states. A number of weaker bands are observed, and assigned to pyridine ring modes. These modes gain significantly in intensity, while the exocyclic modes diminish, when the spectra are excited at 266 nm (YAG laser, fourth harmonic) in resonance with ring-localized electronic transitions.     

Light-induced chloroplast rearrangements and their action spectra as measured by absorption spectrophotometry

Summary A new combination technique of using both dual-wavelength and opalglas methods for scanning translucent biological samples was applied to leaves of terrestrial plants in order to observe their absorption changes by irradiation and the action spectra for the absorption changes. The measurements of true absorption, free from various effects of scattering, by this technique showed an increase of absorption by weak blue light and a decrease of absorption by strong blue light for almost all of the leaves of 20 plant species examined. These weak- and strong-light responses in absorption were reversible. The fractional increase and decrease of absorbance at 678 nm by weak and strong light were highest, +20% and -31%, for leaves of Begonia semperflorens Link et Otto, and +12% and -13% for leaves of foxtail, Setaria viridis (L.) Beauv., the species examined in further experiments. The response to strong light proceeded to completion earlier than did that to weak light. The strong-light response could be observed separately from the weak-light response by using a leaf pre-irradiated with weak blue light. The responses were measured as a function of light intensity by scanning a single leaf irradiated locally at different intensities, and the action spectra for these responses were measured by scanning a leaf irradiated locally at different wavelengths but at identical intensities. The action spectra for these opposite responses were similar, and showed a band at 450 nm with shoulders but no band in the red region. Microscopic observations of chloroplasts in leaves during irradiation indicated that these changes in absorption are mostly due to rearrangements of chloroplasts in cells caused by irradiation.     

Behavior of red king crab larvae: phototaxis, geotaxis and rheotaxis

Zoeae of Paralithodes camtschatica were positively phototactic to white light intensities above 1 x 10(13) q cm-2 s-1. Negative phototaxis occurred at low (1 x 10(12) q cm-2 s-1), but not high intensities (2.2 x 10(16) q cm-2 s-1). Phototactic response was directly related to light intensity. Zoeae also responded to red, green and blue light. Zoeae were negatively geotactic, but geotaxis was dominated by phototaxis. Horizontal swimming speed of stage 1 zoeae < 4 d old was 2.4 +/- 0.1 (SE) cm s-1 and decreased to 1.7 +/- 0.1 cm s-1 in older zoeae (P < 0.01). Horizontal swimming speed of stage 2 zoeae was not significantly different from > or = 4 d old stage 1 zoeae. Vertical swimming speed, 1.6 +/- 0.1 cm s-1, and sinking rate, 0.7 +/- 0.1 cm s-1, did not change with ontogeny. King crab zoeae were positively rheotactic and maintained position in horizontal currents less than 1.4 cm s-1. Starvation reduced swimming and sinking rates and phototactic response.     

柚树(Citrus grandis)叶片光合作用对补增UV-B辐射的响应

生长在人工光照 4 0 0μmol m- 2 s- 1 下的柚树幼树光合速率的最大值为 1 0 .2± 0 .5μmol m- 2 s- 1 ;而补增UV-B辐射 ( 3.8-4 .2μW cm- 2 ,2 4 5～ 2 97nm,4 5d)的叶片则为 6.4± 0 .8μmol m- 2 s- 1 ,较对照植株降低37.2 %。对照植物的表观量子产率 (固定 mol CO2 mol- 1量子 )为 0 .0 75± 0 .0 1 2 ,而经 UV-B辐射处理植株则为0 .0 4 1± 0 .0 0 8,明显较对照植株低。UV-B辐射处理使植株叶片的光呼吸和不包括光呼吸的 CO2 补偿点增高。对照植株叶片的最大值的 CO2 羧化速率 (μmol m- 2 s- 1 )为 57.1± 1 .5μmol m- 2 s- 1 ,较 UV-B辐射处理的高30 .9% ,而 UV-B辐射处理的植株的光合电子传递速率较对照低 30 %。同时 UV-B辐射植株叶片有较低的光能转化效率 ,其较对照低 39.1 % ,叶片亦含有较低的叶绿素含量。结果表明 ,UV-B辐射明显抑制叶片光合羧化速率和光合电子传递速率 ,UV-B辐射可能抑制包括 Rubisco羧化作用在内的多个光合生理过程 ,降低叶片光合速率。柚树叶片对 UV-B辐射敏感 ,选育抗 UV-B辐射的柚树品种势在必行。     

Blue Light Induction of Carbonic Anhydrase Activity in Chlamydomonas reinhardtii

Blue light was specifically required for the induction of carbonicanhydrase (CA) activity in Chlamydomonas reinhardtii. The enhancingeffect of blue light (460 nm) was saturated at energy fluencerate as low as 0.6-0.8 W/m². The wavelength dependency curvehad a peak at 460 nm with no effect at wavelengths above 510nm, thus showing the strong similarities to other blue lightresponses in microalgae. CA induction was strongly inhibitedby UV irradiation at 280 nm. Experiments with the flavin quencher,potassium iodide, suggested that flavin is somehow involvedin CA induction. ¹On leave from the Institute of Biological Sciences, Collegeof Arts and Sciences, University of the Philippines at Los Banos,4031 College, Laguna, Philippines. (Received August 29, 1988; Accepted November 26, 1988)     

The soluble cytochromes c of methanol-grown Hyphomicrobium X. Evidence against the involvement of autoreduction in electron-acceptor functioning of cytochrome cL.

Hyphomicrobium X, grown on methanol with O2 or nitrate as electron acceptor, contains two major soluble cytochromes c. These were isolated in electrophoretically homogeneous form. They are related to cytochromes c already described for other methylotrophic bacteria and designated cytochromes cH and cL (properties indicated in that order) in view of the following characteristics: absorption maxima of the reduced forms (414, 520 and 551 nm and 414, 520 and 550 nm); molar absorption coefficients of the alpha-bands (23,700 M-1.cm-1 and 21,600 M-1.cm-1); maxima of the alpha-bands (no splitting) at 77 K (547.6 nm and 548.5 nm); Mr values of the native proteins (15,000 and 19,500); pI values (7.4 and 7.5, and 4.3); midpoint potentials at pH 7.0 (+292 mV and +270 mV). Both were monomers containing 1 haem c group per protein molecule, the oxidized forms binding cyanide at high pH. Autoreduction also occurred at high pH but at a rate significantly lower than that reported for other ferricytochromes c. On the other hand, the reverse situation applies to the reduction of ferricytochrome cL by reduced methanol dehydrogenase, the reduction occurring instantaneously at pH 7 but much more slowly at pH 9 (ferricytochrome cH was reduced at a 7-fold lower rate, but the rates at pH 7 and 9 were similar). Insignificant reduction was observed with cyclopropanol-inactivated enzyme or with enzyme in the presence of EDTA. In view of the dissimilarities, it is concluded that different mechanisms operate in the autoreduction of ferricytochrome cL and in its reduction by reduced methanol dehydrogenase.     

M?ssbauer, EPR, and optical studies of the P-460 center of hydroxylamine oxidoreductase from Nitrosomonas. A ferrous heme with an unusually large quadrupole splitting

Hydroxylamine oxidoreductase from Nitrosomonas europeae catalyzes the oxidative conversion of NH2OH to NO-2. The enzyme, Mr = 220,000, has an (alpha beta)3 subunit structure with each alpha beta subunit containing 7-8 c-type hemes and one unusual prosthetic group, termed P-460. The P-460 is also found in a Mr approximately equal to 17,000 protein (P-460 fragment). M?ssbauer spectra of the reduced P-460 groups, in hydroxylamine oxidoreductase and the fragment, exhibit nearly identical quadrupole doublets with an unusually large splitting, delta EQ = 4.21 mm/s (no ferrous heme protein is known with delta EQ greater than 2.75 mm/s). The observed isomer shift, delta = 0.96 mm/s at 4.2 K, shows that the P-460 iron is high spin ferrous. Treatment of oxidized hydroxylamine oxidoreductase with H2O2 followed by reduction or exposure of the native sample to CO led to the disappearance of both the characteristic 460 nm absorption band (epsilon = 89 mM-1 cm-1) and the delta EQ = 4.21 mm/s doublet. The iron of the oxidized P-460 fragment is high spin ferric, with M?ssbauer and EPR parameters very similar to those of metmyoglobin. Optical spectra of the reduced P-460 fragment show long wavelength bands at 650 and 688 nm which are sensitive to treatment of the fragment with reagents which react with P-460. These bands were, however, not detected in hydroxylamine oxidoreductase. The spectroscopic and chemical evidence obtained to date suggests strongly that the P-460 iron resides in a heme-like macrocycle although the presumed porphyrin must have some unusual features.     

Purification, characterization and localization of serine protease of Morris hepatoma 8999.

1. A serine protease of hepatoma 8999, isolated in the mitochondrial fraction, was purified and crystallized. The purified enzyme was apparently homogeneous on ultracentrifugal analysis and polyacrylamide disc gel electrophoresis. The ratio of absorbance at 280 nm and 260 nm, A280/A260, was 1.90 and its absorption coefficient, A280 1% was 10.5 cm-1 estimated from dry weight measurements. Its S20, w value was 2.23 S and its molecular weight was estimated to be 24000 +/- 1000. The enzyme contained twice as much lysine, arginine and histidine as chymotrypsinogen did, but had a very similar amino acid composition to serine protease from skeletal muscle. Its isoelectric point was pH 10.6. 2. The substrate specificity of the enzyme was the same as that of chymotrypsin A. Its Km and kcat values for N-acetyl-L-tyrosine ethyl ester, N-acetyl-L-phenylalanine ethyl ester and N-acetyl-L-tryptophan ethyl ester were 0.35 mM and 10.69 s-1, 0.38 mM and 10.7 s-1, and 0.11 mM and 11.8 s-1, respectively. Its activity was completely inhibited by phenylmethylsulfonyl fluoride and partially inhibited with tosylphenylalanine chloromethyl ketone. 3. The enzyme was shown to be located in different granules from the intracellular particules (light and heavy mitochondrial fraction) by sucrose density gradient centrifugation, and it was stained in mast cells of the hepatoma 8999 by the immunofluorescent technique. 4. Serine protease is present in different amounts in various organs of rat and the enzyme from hepatoma 8999 gave a single band that fused completely with those of the enzymes from skeletal muscle, heart, liver and kidney, respectively, on Ouchterlony double-diffusion analysis using antiserum to the crystalline enzyme of hepatoma 8999, but the enzyme from small intestine did not react with the antiserum.     

Bertalanffy-like fluorescence staining with 3-dimethylamino-6-methoxyacridine

Three new acridine dyes, 3-dimethylamino-6-methoxyacridine 1, 3-amino-6-methoxyacridine 2 and 3-amino-7-methoxyacridine 3, have been prepared and tested as fluorochromes of LM- and HeLa-cells. The dyes are basic compounds (pKA: 1 8,76; 2 8,01; 3 7,65) and form cations in neutral or acidic aqueous solutions by addition of a proton to the aza-nitrogen atom of the heterocycle. The fluorochromes stain fixed LM- and HeLa-cells at pH = 6. The fluorescence shows metachromasy similar to the staining with acridine orange AO according to the technique of Bertalanffy. But there is less fading of the fluorescence. The dye 1 is the most suitable fluorochrome of the series. It was studied in detail. Using optimized staining conditions the fluorescence of the nucleus is yellow-green that of the cytoplasm and the nucleoli orange or brownish-red. Enzymatic digestion experiments show that the dye cations are bound to DNA in the nucleus and to RNA in the cytoplasm or nucleoli. The absorption and emission spectra of the stained cells have been studied by means of microspectrophotometry. The absorption spectra of the nucleus and the cytoplasm are very similar. The maximum of the long wave length absorption of both occurs at 21400 cm-1 (467 nm) with a shoulder at ca 20100 cm-1 (498 nm). The fluorescence spectra of nucleus and cytoplasm of metachromatically stained cells are different. The emission maximum of the cytoplasm and nucleoli, 16200 cm-1 (617 nm), is red-shifted relative to the maximum of the nucleus, 18200 cm-1 (549 nm). This shift causes the metachromatic fluorescence effect. In addition we studied the concentration dependence of the absorption and fluorescence spectra of the cation 1 in aqueous solution, pH = 6, in the concentration range 6 X 10(-6)-6 X 10(-4) M. Shape and maximum of the long wave length absorption and emission depend only slightly on the concentration: Mean value of absorption maximum ca 21500 cm-1 (465 nm), shoulder at ca 20300 cm-1 (493 nm), fluorescence maximum ca 18300 cm-1 (547 nm). With growing concentration diminishes the molar absorptivity. This decrease in absorptivity and isosbestic points in the absorption spectra indicate the formation of dimers with growing dye concentration. The absorption spectra of the metachromatically stained cells and of the dye in aqueous solution are very similar.(ABSTRACT TRUNCATED AT 400 WORDS)     

Action Spectrum for the Timing of Photo-Induced Cell Division in Adiantum Gametophytes

The photo-induced cell division in single-celled protonemata of the fern Adiantum capillus-veneris was studied. When the protonemata were exposed to monochromatic light at 50 nm intervals between 350 and 750 nm, irradiations in the blue and near-ultraviolet regions effectively induced cell division, while wavelengths longer than 550 nm showed no such effect. As reciprocity between duration and intensity was observed within the range of incident energy used, the action spectrum for the frequency of the photo-induced cell divisions 24 h after irradiation was determined between 360 and 510 nm at 10 nm intervals. Furthermore, the previously known effect of phytochrome on the timing of the cell division was minimized by a short exposure to red light given immediately after the monochromatic irradiation. The resulting action spectra showed a peak in the neighborhood of 460 nm with shoulders and another peak in the near-ultraviolet region.     

Contribution of the resonance Raman spectroscopy to the identification of Z DNA

Poly(dG-dC).poly(dG-dC) at low salt concentration (0.1 M NaCl) and at high salt concentration (4.5 M NaCl) has been studied by Raman resonance spectroscopy using two excitation wavelengths: 257 nm and 295 nm. As resonance enhances the intensity of the lines in a proportion corresponding to the square of the molar absorption coefficient, the intensities of the lines with 295 nm wavelength excitation are enhanced about sevenfold during the B to Z transition. With 257 nm excitation wavelength the 1580 cm-1 line of guanosine is greatly enhanced in the Z form whereas with 295 nm excitation several lines are sensitive to the modifications of the conformation: the guanine band around 650 cm-1 and at 1193 cm-1 and the bands of the cytosines at 780 cm-1, 1242 cm-1 and 1268 cm-1. By comparison with the U.V. resonance Raman spectra of DNA, we conclude that resonance Raman spectroscopy allows one to characterize the B to Z transition from one line with 257 nm excitation wavelength and from three lines with 295 nm excitation. The conjoined study of these four lines should permit to observe a few base pairs being in Z form in a DNA.     

Effects of growth inhibitors and ultraviolet irradiation on F pili

The effects of chloramphenicol, nalidixic acid, mitomycin C, NaCN, and ultraviolet irradiation at 253.7 nm on F pili production by Escherichia coli cells was studied by electron microscopy. The results show that cells contain pools of pili protein, and that assembly does not require synthesis of protein or deoxyribonucleic acid (DNA). NaCN (2 x 10(-2)m) prevents the reappearance of pili and causes existing pili to disappear quickly from the cell surface. This suggests that energy is used in the assembly of pili and to retain pili on the cell. Cells irradiated with high doses (10(4)ergs/mm(2)) of 253. 7 nm light produce fewer pili, and these are shorter than normal. Dose-response curves for number of pili per cell and length of pili resemble single hit kinetics, showing 37% survival at 10(4) ergs/mm(2) and 2 x 10(4) ergs/mm(2), respectively. This suggests that DNA is at the site where pili are produced, and that it may be involved in the assembly of pili.     

Electron transport components involved in hydrogen oxidation in free-living Rhizobium japonicum.

Membranes from free-living Rhizobium japonicum were isolated to study electron transport components involved in H2 oxidation. The H2/O2 uptake rate ratio in membranes was approximately 2. The electron transport inhibitors antimycin A, cyanide, azide, hydroxylamine, and 2-n-heptyl-4-hydroxyquinoline-N-oxide (HQNO) inhibited H2 uptake and H2-dependent O2 uptake significantly. H2-reduced minus O2-oxidized absorption difference spectra revealed peaks at 551.5, 560, and 603 nm, indicating the involvement of cytochromes c, b, and a-a3, respectively. H2-dependent cytochrome reduction was completely inhibited in the presence of 0.15 mM HQNO. This inhibition was relieved by the addition of 0.1 mM menadione. Evidence is presented for the involvement of two b-type cytochromes in H2 oxidation. One b-type cytochrome was not reduced by ascorbate and had an absorption peak at 560 nm. The reduction of this cytochrome by H2 was not inhibited by cyanide. A second b-type cytochrome, cytochrome b', was not reduced by H2 in the presence of cyanide. This cytochrome had an absorption peak at 558 nm. Carbon monoxide difference spectra with H2 as reductant provided evidence for the involvement of cytochrome o as well as cytochrome a3 in H2 oxidation. H2 uptake activity in cell-free extracts was inhibited by UV light irradiation. Most of the activity of the UV-treated extracts was restored with the addition of ubiquinone. The restored activity was inhibited by cyanide. A branched electron transport pathway from H2 to O2 is proposed.     

Interaction of glutathione reductase with heavy metal: the binding of Hg(II) or Cd(II) to the reduced enzyme affects both the redox dithiol pair and the flavin

To determine the inhibition mechanism of yeast glutathione reductase (GR) by heavy metal, we have compared the electronic absorption and resonance Raman (RR) spectra of the enzyme in its oxidized (Eox) and two-electron reduced (EH2) forms, in the absence and the presence of Hg(II) or Cd(II). The spectral data clearly show a redox dependence of the metal binding. The metal ions do not affect the absorption and RR spectra of Eox. On the contrary, the EH2 spectra, generated by addition of NADPH, are strongly modified by the presence of heavy metal. The absorption changes of EH2 are metal-dependent. On the one hand, the main flavin band observed at 450 nm for EH2 is red-shifted at 455 nm for the EH2-Hg(II) complex and at 451 nm for the EH2-Cd(II) complex. On the other hand, the characteristic charge-transfer (CT) band at 540 nm is quenched upon metal binding to EH2. In NADPH excess, a new CT band is observed at 610 nm for the EH2-Hg(II)-NADPH complex and at 590 nm for EH2-Cd(II)-NADPH. The RR spectra of the EH2-metal complexes are not sensitive to the NADPH concentration. With reference to the RR spectra of EH2 in which the frequencies of bands II and III were observed at 1582 and 1547 cm-1, respectively, those of the EH2-metal complexes are detected at 1577 and 1542 cm-1, indicating an increased flavin bending upon metal coordination to EH2. From the frequency shifts of band III, a concomitant weakening of the H-bonding state of the N5 atom is also deduced. Taking into account the different chemical properties of Hg(II) and Cd(II), the coordination number of the bound metal ion was deduced to be different in GR. A mechanism of the GR inhibition is proposed. It proceeds primarily by a specific binding of the metal to the redox thiol/thiolate pair and the catalytic histidine of EH2. The bound metal ion then acts on the bending of the isoalloxazine ring of FAD as well as on the hydrophobicity of its microenvironment.     

Photochemical revelation of the stacking aggregation of thymine chromophores in water solutions of polythymidylic acid

The structure heterogeneity of water solutions of polyribothymidylic acid at T(room) was studied from changes caused in their absorption spectra by the photodimerization reaction. Three fractions of thymine chromophores were revealed from the differential absorption spectra: (a) the main fraction consisting of weakly interacting (isolated chromophores) chromophores with the absorption spectrum maximum at approximately 270 nm; (b) pair chromophores of the first type with the absorption spectrum maxima at 260 and 290 nm (exciton splitting 4000 cm(-1)); and (c) pair chromophores of the second type with the absorption spectrum maxima at 250 and 280 nm (exciton splitting 4300 cm(-1)). The revealed aggregates have a relatively high photochemical activity in the photodimerization reaction in comparison with the isolated chromophores. They contribute little to the total absorption spectrum of solutions but make a great contribution to its changes at the initial stages of the UV irradiation of solutions.