INACTIVATION OF Trypanosoma cruzi TRYPOMASTIGOTE FORMS IN BLOOD COMPONENTS BY PHOTODYNAMIC TREATMENT WITH PHTHALOCYANINES

Abstract— -Three phthalocyanine dyes HOSiPcOSi(CH₃)₂(CH₂)₃N(CH₃)₂ (Pc 4), HOSiPc-OSi(CH₃)₂(CH₂)₃N+(CH₃)3I- (Pc 5) and aluminum tetrasulfophthalocyanine hydroxide (AlOHPcS₄) were evaluated for their ability to inactivate the trypomastigote form of Trypanosoma cruzi in fresh frozen plasma (FFP) and red blood cell concentrates (RBCC). The compound Pc 4 was found to be highly effective in killing T. cruzi, Pc 5 less effective and AlOHPcS₄ ineffective. With FFP as the medium, a complete loss of parasite infectivity in vitro (≥5 log₁₀) was found to occur with 2 μ M Pc 4 after irradiation with red light (>600 nm) at a fiuence of 7.5 J/cm², while with RBCC as the medium, a complete loss was found to occur at a fiuence of 15 J/cm². Even without illumination, Pc 4 at 2 μ M also killed about 3.7-4.1 log₁₀ of T. cruzi in FFP during 30 min. Observed differences in T. cruzi killing by the various phthalocyanines may relate to differences in binding; Pc 4 binds to the parasites about twice as much as Pc 5. Ultrastructural analysis of treated parasites suggests that mitochondria are a primary target of this photodynamic treatment. The data indicate that Pc 4 combined with exposure to red light could be used to eliminate bloodborne T. cruzi parasites from blood components intended for transfusion. The inactivation of T. cruzi by Pc 4 in the dark suggests a possible therapeutic application.     

HEMATOPORPHYRIN PHOTOSENSITIZATION OF SERUM ALBUMIN and SUBTILISIN BPN'

—The photosensitized inactivation of subtilisin BPN' by free hematoporphyrin (HP) followed exponential kinetics with positive mechanistic tests for the involvement of singlet oxygen (¹O₂) as the principal intermediate. The photoinactivation quantum yield was 0.029 at 390 nm in oxygen-saturated, D₂O buffer at pH 7.0. The effects of HP binding were investigated for tryptophan oxidation in bovine serum albumin (BSA) and human serum albumin (HSA) at high protein:HP concentration ratios where the HP was > 97% complexed. The reaction kinetics were non-exponential and mimick a second-order process in the initial stages. The rate of HP photobleaching was 30-fold faster for complexed HP compared with free HP, which was shown to account for the observed kinetics. Mechanistic tests showed that ¹O₂ was the dominant photooxidizing intermediate of tryptophan residues and that it was not involved in the accompanying photobleaching of HP. The quantum yield for tryptophan oxidation in BSA was 0.11 at 390 nm in oxygen-saturated, D₂O buffer at pH 8.0. The reactivity of HSA was approximately 2-fold lower than BSA for equivalent conditions. Estimates of the reaction cross sections led to 3 Ų for the inactivation of subtilisin BPN' by ¹O₂ and 20 Ų for the oxidation of tryptophan in BSA.     

Illumination with Ultraviolet or Visible Light Induces Chemical Changes in the Water-soluble Manganese Complex, [Mn4O6(bpea)4]Br4

We measured the photosensitivity of an artificial tetranuclear oxo–Mn(IV) complex, [Mn₄O₆(bpea)₄]Br₄, which has an adamantane-shaped {Mn₄O₆}⁴⁺ core. Illumination caused changes in the absorption spectrum of the compound consistent with a one-electron reduction in the compound. Bromide appears to be the most probable electron donor in the reaction system. Chemical modification of the cluster appears to destabilize it, predisposing it to reductive degradation. UV light was more efficient than visible light in causing the changes. The data support the suggestion that the natural oxygen-evolving Mn complex is photosensitive and can oxidize components of the oxygen-evolving complex in its excited state causing photoinhibition, and that photostability is an important issue in designing Mn complexes for artificial photosynthesis. Furthermore, light-induced oxidation of bromide by [Mn₄O₆(bpea)₄]⁴⁺ may suggest that oxidation of chloride is involved in natural water splitting or has been involved during the evolution of the water-splitting enzyme.     

Study of the factors affecting the photoelectrode characteristics of a perylene/phthalocyanine bilayer working in the water phase

Some types of phthalocyanines (MPc (M = H(2), Cu, or Zn), a p-type semiconductor) were used in combination with 3,4,9,10-perylenetetracarboxyl-bisbenzimidazole (PTCBI, an n-type semiconductor), with which those photoelectrode characteristics in the water phase were investigated in terms of kinetics. Each film of the PTCBI/MPc bilayer functioned as a photoanode, where the photoinduced oxidation of thiol occurs at the MPc/water interface along with the hole conduction through the MPc layer. The holes originate on account of the photophysical events in the p/n interior, involving the charge separation of excitons at the p/n interface. The typical photoelectrochemical characteristic in the PTCBI/MPc photoanodes involved a transient photocurrent occurring in the initial stage under illumination (under potentiostatic conditions): thereafter, it attained a steady state. Moreover, both the initial spiky photocurrents and the steady-state photocurrents exhibited saturation at higher concentrations. An analysis with photoelectrode kinetics was performed by assuming an adsorption step prior to a rate-limiting charge transfer step, where equations were applied to photocurrents based on the Langmuir adsorption equilibrium. The kinetic analyses evidently showed that the photoanodic reactions are kinetically dominated by the charge transfer between MPc and thiol, where the overall kinetics for thiol oxidation decreases in the following order: H(2)Pc > ZnPc > CuPc; that is, it appeared that H(2)Pc acts as the more efficient photofunctional interface capable of oxidation in the water phase when PTCBI was concurrently employed as an electron conductor. Considering that the photocurrent generated is proportional to the surface concentration of thiol (Gamma) at the MPc as well as the intrinsic oxidation rate (cf., ZnPc > H(2)Pc approximately CuPc), the higher efficiency in the output at the H(2)Pc surface was attributed to an exceptionally high Gamma (i.e., from the kinetic analyses, the Gamma value at the H(2)Pc surface was also inferred to be 2-3 times higher than that at the other MPcs). Through the present kinetic analysis, it also revealed that the activity for thiol oxidation taking place at Pc ring is comparable to that at the conventional active catalysts (i.e., polycarboxyphthalocyaninato Co(ii) and Fe(iii)) where a central metal is an active site.     

STOICHIOMETRY and KINETICS OF PROTON RELEASE UPON PHOTOSYNTHETIC WATER OXIDATION

Abstract— We studied the magnitude and the rise kinetics of proton release into the interior of thylakoids by flash spectrophotometty with neutral red as pH indicator. Excitation of dark-adapted thylakoids by a series of between 4 and 11 flashes produced a complex pattern of proton release into the thylakoid lumen. Proton release upon each flash was time resolved.
A slow component of proton release oscillated weakly in magnitude with period of two as function of flash number. It exhibited a half-rise time of approximately 20 ms from the very first flash on, and it was abolished by inhibitors of plastohydroquinone oxidation. This component was attributed to the oxidation of plastohydroquinone by PS I via the Cytb₆/f complex.
Additionally, rapid and multiphasic proton release was observed with half-rise times of less than 2 ms which exhibited a pronounced and damped oscillation with period of four as function of flash number. This rapid proton release was attributed to water oxidation. A detailed kinetic analysis suggested that proton release occurred with the following stoichiometry and with the following half-rise times during the transitions S₁ S_i+1 of water oxidation: 1 H⁺(250 μs, S₀→₁): 0 H⁺(S₁→ S₂):1 H⁺(200 μs, S₂→S₃):2 H⁺(1.2 ms, S₃→ S₄→ S₀) . Proton release and proton rebinding upon oxidation and reduction of the intermediate electron carrier Z, respectively, may have influenced the kinetics of the respective proton yields but not the stoichiometric pattern.     

PHOTOINACTIVATION OF A Chlamydomonas MUTANT(NL–11) IN THE PRESENCE OF METHIONINE: ROLES OF H2O2 and O-2

Abstract— A mutant of Chlamydomonas reinhardtii (NL–11) isolated from a wild type (137c+) was inactivated in the light in the presence of methionine at concentrations where the wild type was not inactivated. The inactivation was suppressed by either catalase or superoxide dismutase (SOD). Light-induced H₂O₂ formation and nitroblue tetrazolium (NBT) reduction inNL–11 were greater than those in the wild type. Methionine stimulated both the H₂O₂ formation and the NBT reduction inNL–11 as well as the wild type. The light-induced NBT reduction inNL–11 in the presence of methionine was partially suppressed by externally added SOD suggesting the participation of O^-₂. These results suggest that the hypersensitivity ofNL–11 to methionine in the light is due to stimulated formation of H₂O₂ and O^-₂.     

SPECTRAL FEATURES OF THE BOUND ELECTRON ACCEPTOR A2 OF PHOTOSYSTEM I

Abstract— Difference spectrum for the reduction of A₂, a bound secondary electron acceptor of photo-system I, in the thylakoid membranes of a thermophilic blue-green alga, Synechococcus sp., was determined by subtracting the difference spectrum of P700 photooxidation from the difference spectrum for flash-induced absorption changes due to oxidation of P700 and reduction of A₂, or by measuring light-induced absorption changes under reducing conditions where reduced A₂ accumulates. The spectrum showing a broad bleaching with two maxima at 420 and 440 nm indicates that A₂ is an iron-sulfur center different from P430.     

LIGHT-DRIVEN H2 PRODUCTION WITH PROFLAVIN AND HYDROGENASE: COMPARISON OF CYTOCHROME C3 AND METHYL VIOLOGEN AS e- MEDIATORS

We have studied the hydrogenase-catalyzed production of H, when either its natural electron mediator cytochrome c₃ (c₃) or the artificial mediator methyl viologen (MV) was reduced by illumination of proflavin (PF) in the presence of ethylenediaminetetraacetate (EDTA). The reduction rates of MV and c₃ were comparable at equivalent concentrations of PF, taking into account the four redox sites of c₃. However, when the concentration of c₃ exceeded that of PF, the reduction rate decreased. We explain this by light quenching. In the H₂-producing system, MV was more efficient than c₃ as electron mediator when the initial reaction rates were compared. However, under certain conditions with MV, the rate of H₂ production decreased rapidly with time of illumination, whereas with c₃ it consistently remained stable. Possible reasons for this difference are discussed.
Severe inactivation of hydrogenase was observed in the absence of the primary electron donor EDTA. It is concluded that this inactivation is caused by the excited state of PF     

PHOTODYNAMIC EFFECTS OF NEW SILICON PHTHALOCYANINES: In vitro STUDIES UTILIZING RAT HEPATIC MICROSOMES AND HUMAN ERYTHROCYTE GHOSTS AS MODEL MEMBRANE SOURCES

Abstract— Photodynamic therapy (PDT) of cancer is a modality that relies upon the irradiation of tumors with visible light following selective uptake of a photosensitizer by the tumor tissue. There is considerable emphasis to define new photosensitizers suitable for PDT of cancer. In this study we evaluated six phthalocyanines (Pc) for their photodynamic effects utilizing rat hepatic microsomes and human erythrocyte ghosts as model membrane sources. Of the newly synthesized Pc, two showed significant destruction of cytochrome P-450 and monooxygenase activities, and enhancement of lipid peroxidation, when added to microsomal suspension followed by irradiation with ∼ 675 nm light. These two Pc named SiPc IV (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₂) and SiPc V (HOSiPcOSi[CH₃]₂[CH₂]₃N[CH₃]₃¹ I) showed dose-dependent photodestruction of cytochrome P-450 and monooxygenase activities in liver microsomes, and photoenhancement of lipid peroxidation, lipid hydroperoxide formation and lipid fluorescence in rnicrosomes and erythrocyte ghosts. Compared to chloroaluminum phthalocyanine tetrasulfonate, SiPc IV and SiPc V produced far more pronounced photodynamic effects. Sodium azide, histidine, and 2,5-dimethylfuran, the quenchers of singlet oxygen, afforded highly significant protection against SiPc IV- and SiPc V-mediated photodynamic effects. However, to a lesser extent, the quenchers of superoxide anion, hydrogen peroxide and hydroxyl radical also showed some protective effects. These results suggest that SiPc IV and SiPc V may be promising photosensitizers for the PDT of cancer.     

ELIMINATION OF THE EFFECT OF CONTAMINATING CO2 IN THE 18O-LABELING OF THE CO2 PRODUCED IN BIOLUMINESCENT REACTIONS

Abstract— Although the mechanism of bioluminescent reactions in various species, such as fireflies, ostracod crustaceans ( Cypridina ), sea pansies ( Renilla ), and the deep-sea shrimp Oplophorus , are thought to involve dioxetanone intermediates, studies reported in the past from different laboratories have included widely different experimental results, most likely due to various factors including the effects of contaminating CO₂. With the improved technique employed in the present study, bioluminescent reactions of the firefly and Cypridina in ¹⁸O₂ gas resulted in an incorporation of over 75% of ¹⁸O into one oxygen of the product CO₂. with a reproducibility within a few per cent. When ¹³CO₂. instead of the product CO₂ of the bioluminescent reaction, was studied in an H₂¹⁸O medium, the exchange of one oxygen of ¹³CO₂ with H₂O was 64%. and the effect of contaminant CO₂ amounted to 1418% of the total CO₂. These results suggest that every molecule of CO₂ formed in the bioluminescent reactions of the firefly and Cypridina had intially contained 1 oxygen atom derived from O₂.     

Biomaterial engineered electrodes for bioelectronics

A series of single-cysteine-containing cytochrome c, Cyt c, heme proteins including the wild-type Cyt c (from Saccharomyces cerevisiae) and the mutants (V33C, Q21C, R18C, G1C, K9C and K4C) exhibit direct electrical contact with Au-electrodes upon covalent attachment to a maleimide monolayer associated with the electrode. With the G1C-Cyt c mutant, which includes the cysteine residue in the polypeptide chain at position 1, the potential-induced switchable control of the interfacial electron transfer was observed. This heme protein includes a positively charged protein periphery that surrounds the attachment site and faces the electrode surface. Biasing of the electrode at a negative potential (-0.3 V vs. SCE) attracts the reduced Fe(II)-Cyt c heme protein to the electrode surface. Upon the application of a double-potential-step chronoamperometric signal onto the electrode, where the electrode potential is switched to +0.3 V and back to -0.3 V, the kinetics of the transient cathodic current, corresponding to the re-reduction of the Fe(III)-Cyt c, is controlled by the time interval between the oxidative and reductive potential steps. While a short time interval results in a rapid interfacial electron-transfer, ket1 = 20 s-1, long time intervals lead to a slow interfacial electron transfer to the Fe(III)-Cyt c, ket2 = 1.5 s-1. The fast interfacial electron-transfer rate-constant is attributed to the reduction of the surface-attracted Fe(III)-Cyt c. The slow interfacial electron-transfer rate constant is attributed to the electrostatic repulsion of the positively charged Cyt c from the electrode surface, resulting in long-range electron transfer exhibiting a lower rate constant. At intermediate time intervals between the oxidative and reductive steps, two populations of Cyt c, consisting of surface-attracted and surface-repelled heme proteins, are observed. Crosslinking of a layered affinity complex between the Cyt c and cytochrome oxidase, COx, on an Au-electrode yields an electrically-contacted, integrated, electrode for the four-electron reduction of O2 to water. Kinetic analysis reveals that the rate-limiting step in the bioelectrocatalytic reduction of O2 by the integrated Cyt c/COx electrode is the primary electron transfer from the electrode support to the Cyt c units.     

CHLOROPHYLL PHOTOSENSITIZED ELECTRON TRANSFER IN PHOSPHOLIPID BILAYER VESICLE SYSTEMS: SECONDARY OXIDATION OF REDUCED VIOLOGEN ACCEPTORS BY Ru(NH3)63+

Abstract— The kinetics of the oxidation of a homologous series of 4,4'-di(n-alkyl)-bipyridinium (viologen) radicals by Ru(NH₃)₆³⁺ in vesicle suspensions was studied using laser flash photolysis. The viologen radicals were produced photochemically in the bilayer membrane phase of the vesicles by electron transfer from the triplet state of chlorophyll-α. At high concentrations of Ru(NH₃)₆³⁺, the rate of oxidation of the viologen radicals in the aqueous phase was limited by the rate at which the radicals diffused from the membrane to the aqueous phase. The exit rate constant decreased from 2 × 10⁵ s⁻¹ for the methyl viologen radical to 4 × 10³ s⁻¹ for the pentyl viologen radical. Both the exit rate constants and the calculated values for the equilibrium association constants of the viologen radicals were unexpectedly insensitive to the length of their alkyl substituents. This, as well as other data, suggests that the radicals that diffused into the aqueous phase tended to remain associated with the membrane-water interface.     

AMPHIPHILIC COPOLYMERS AS MEDIA FOR LIGHT-INDUCED ELECTRON TRANSFER-II. ELECTROSTATIC EFFECT ON THE BACK ELECTRON TRANSFER

Abstract— Photosensitized reduction of zwitterionic viologen (SPV) and methyl viologen (MV²⁺) was investigated using an amphiliphilic copolymer having phenanthryl and sulfonate groups (APh) as photosensitizer in aqueous solutions. In the presence of triethanolamine the accumulation of SPV * (photoproduct) was found to be faster than that of MV⁺. This attributed to the electrostatic repulsion between SPV. and anionic segments of APh. Such difference between SPV and MV²⁺ was minimized in the case of the related monomer model. Retardation of the back reaction for the APh-SPV system was also demonstrated by laser photolysis, k _b= 8.7 × 10⁷ M ^-1 s^-1 for the polymer system as compared to k _b= 2.8 × 10⁹ M ^-1 s^-1 for the monomer model system. Strong salt-effects on the yield of the photoreduction and the rate of back reaction confirm the strong electrostatic interaction between the photoproducts and polyanions. This remarkable electrostatic effect of the polyanions was simulated by electrochemical redox reactions by using a graphite electrode coated with APh.     

THE EFFECT OF IRRADIANCE ON VIRUS STERILIZATION AND PHOTODYNAMIC DAMAGE IN RED BLOOD CELLS SENSITIZED BY PHTHALOCYANINES

Abstract— Phthalocyanines are being studied as photosensitizers for virus sterilization of red blood cells (RBC). During optimization of the reaction conditions, we observed a marked effect of the irradiance on production of RBC damage. Using a broad-band light source (600–700 nm) between 5 and 80 mW/ cm², there was an inverse relationship between irradiance and rate of photohemolysis. This effect was observed with aluminum sulfonated phthalocyanine (AlPcS_n) and cationic silicon (HOSiPc-OSi[CH₃]₂ [CH₂]₃N⁺[CH₃]₃I^- phthalocyanine (Pc5) photosensitizers. The same effect occurred when the reduction of RBC negative surface charges was used as an endpoint. Under the same treatment conditions, vesicular stomatitis virus inactivation rate was unaffected by changes in the irradiance. Reduction in oxygen availability for the photochemical reaction at high irradiance could explain the effect. However, theoretical estimates suggest that oxygen depletion is minimal under our conditions. In addition, because the rate of photohemolysis at 80 mW/cm² was not increased when irradiations were carried out under an oxygen atmosphere this seems unlikely. Likewise, formation of singlet oxygen dimoles at high irradiances does not appear to be involved because the effect was unchanged when light exposure was in D₂O. While there is no ready explanation for this irradiance effect, it could be used to increase the safety margin of RBC virucidal treatment by employing exposure at high irradiance, thus minimizing the damage to RBC.     

A PHYTOCHROME STUDY USING TWO-LASER/TWO-COLOR FLASH PHOTOLYSIS: I700 IS A MANDATORY INTERMEDIATE IN THE PrPfr PHOTOTRANSFORMATION

Abstract— The phototransformation of native (124 kDa)oat phytochrome, P_r P_fr, Has been studied at 10C by two laser/ two-color flash photolysis. the overall P_rP_fr reaction yield did not vary with temperature within the range4–21C. Foloeing the excitation of P_r with a single 15 ns laser flash at 650nm, the formation of P_fr was quantitavely measured in a time-resolved experiment in the presence of a second 8 ns laser flash at 710 nm delayed from the initial flash. the second laser flash causes at 1.0 s after the initial laser flash a depletion of the uintermediate I₇₀₀ as welll as a reduction of the P_fr absorption at 730 nm. The depletion of I₇₀₀ correlates quantitavely with the reduction of P_fr formation. The absorpton spectra of I₇₀₀ and of the following intermendiate, I_bi, were calculated assuming that the amount of P_r, which is photoconverted by a single laser, equals the amount of P_fr formed.     

Rapid Tyrosine Phosphorylation of HS1 in the Response of Mouse Lymphoma L5178Y-R Cells to Photodynamic Treatment Sensitized by the Phthalocyanine Pc 4

Abstract— The ability of photodynamic treatment (PDT) with the phthalocyanine Pc 4 to activate cellular signal transduction pathways in murine lymphoma L5178Y-R cells has been assessed by observing increases in protein tyrosine phosphorylation at early times post-PDT. Western blot analysis with an anti-phosphotyrosine antibody revealed a dramatic increase in phosphorylation of two major protein bands of Mr -80000 and -55000 in response to PDT. The increase was PDT dose-dependent, occurred as early as 20 s after initiation of light exposure of Pc 4-pre-loaded cells and was amplified by the presence of the protein tyrosine phosphatase inhibitor, sodium ortho-vanadate (NaV0₄). By immunoprecipitation, one of the Mr –80000 phosphorylated proteins has been identified as HS1, a substrate of nonreceptor-type protein tyrosine kinases. Although vanadate greatly enhanced the level and extent of PDT-induced phosphorylation, it had no influence on overall photocytotoxicity or on the rate of apoptotic DNA fragmentation. Genistein, an inhibitor of protein tyrosine kinases, diminished tyrosine phosphorylation of the Mr –80000 and other proteins and dramatically potentiated cell killing induced by PDT but did not significantly affect PDT-induced apoptosis. The results suggest that PDT rapidly activates a membrane-associated src family kinase(s) in L5178Y-R cells, one substrate of which is HS1, and that protein tyrosine phosphorylation is part of a stress response, protecting a portion of the cells from the lethal effects of PDT but not altering the mechanism by which they die.     

表面带负电荷氨基酸残基的突变对细胞色素b5与细胞色素c结合和识别的影响

利用核磁共振方法研究表面带不同负电荷氨基酸残基突变后的细胞色素b₅与细胞色素c的结合与识别.结果表明,静电作用在细胞色素b₅与细胞色素c的结合过程中有着重要的贡献,而且这些静电贡献在一定程度上具有累加性,E48的贡献略大于E44.同时还证明Browniandynamicssimulations优化出的Glu48-Lys13,Glu56-Lys₈₇,Asp60-Lys86和heme6-propionate-Tml₇₂(细胞色素b₅的残基排在前面)的结合方式在溶液中的确存在.细胞色素b₅突变体(E48,E56/A,D60/A)及[Cr(oxalate)₃]^3-对细胞色素c的表面结合竞争实验表明,细胞色素c表面结合区Site仍然同细胞色素b₅突变体(E48,E56/A,D60/A)有结合作用,只是结合强度上相对于野生细胞色素b₅同细胞色素c的结合有所降低.这表明除上述的Brownian dynamics simulations模型外,尚有其它如Salemme模型等的结合方式,这也揭示出细胞色素b₅和细胞色素c之间的结合是比较动态的.     

PHOTOSYNTHETIC NITRITE REDUCTION BY DITHIOERYTHRITOL AND THE EFFECT OF NITRITE ON ELECTRON TRANSPORT IN ISOLATED CHLOROPLASTS

Abstract. Photosynthetic reduction of nitrite to ammonia with type C chloroplasts from the heterocont alga Bumilleriopsis filiformis was investigated using 3,6-diaminodurene/ascorbate and 3,6-diaminodurene/dithioerythritol (DAD/DTE) as electron donor couple. Rates approach 6–10 μmol NO^-₂ reduced/mg chlorophyll/h and are steady for up to 30 min. The presence of oxygen or NADP⁺ only slightly diminished the rates of nitrite reduction obtained with DAD/DTE. Illuminated chloroplasts reduce oxygen in the presence of DAD/DTE at 135 μmol/mg chlorophyll/h without acceptor supplied. Photosynthetic oxygen uptake by this system in the presence of ferredoxin and NO^-₂, however, is inhibited to 42% by nitrite reductase with concurrent nitrite reduction. NO^-₃ and NO^-₂ have no effect on photosystem I-mediated NADP⁺ reduction, NO^-₂ (10 m M ) inhibits ferricyanide-mediated oxygen evolution to 72%. Also photosystem II reactions assayed e.g. with silicomolybdate are inhibited significantly by NO^-₂ (1 m M ), but only slightly by NO^-₃. Nitrite reductase is inhibited by p -chloromercuribenzoate ( p CMB), and this inhibition is prevented by DTE. Results suggest that photosynthetic nitrite reduction can cope with low concentrations of either compound, provided relevant thiol groups are protected.     

DIFFERENCE SPECTRA OF THE OXIDIZED INTERMEDIATES IN THE PHOTOSYNTHETIC OXYGEN-EVOLVING SYSTEM: EVIDENCE FOR A SMALL S0→S1 ABSORPTION CHANGE

A consensus has emerged in the recent literature on the fact that the UV difference spectrum of the first oxidation step (S₀→ S₁) of the photosynthetic oxygen-evolving complex is significantly different and generally smaller than the spectra of the higher oxidation steps (S₀→ S₁and S₂→ S₃). Discrepancies still persist, however, notably in the 300 nm region where the S₀→ S₁ change was either reported to be markedly smaller than the other changes, or, at variance, to have a similar amplitude. A novel approach is proposed here for estimating the ratio of these changes, requiring no estimate of the Kok model parameters, such as the initial S₀/S₁ratio, or damping coefficients. This was achieved by comparing the absorption difference between two fully deactivated states, differing only in their S₀/S₁, distribution, with the flash-induced changes measured from these states. The results show that, at two wavelengths around 300 nm, the S₀→ S₁ change is at least 4 times, and probably 5–6 times smaller than the S₀→ S₁change.     

HYDROGEN AND OXYGEN PHOTOPRODUCTION BY TITANATE POWDERS

Abstract— Uncoated powders of TiO₂ or SrTiO₃ did not produce H₂ or O₂ on UV irradiation of aqueous suspensions of the powders. TiO₂ powders coated with platinum or rhodium photoproduced H₂ on irradiation (effective wavelengths 334 and 366 nm) and the reaction was stimulated by catalytic quantities of methyl viologen. The turnover numbers for H₂ production relative to TiO₂ were very low suggesting that the powders were not acting catalytically. Hydrogen production was never stoichiometric with respect to TiO₂ and the kinetics of H₂ production were first order, not zero order as would be expected for catalytic photolysis of water. Oxygen was never detected and it appears that H₂ did not arise from water photolysis but rather from oxidation of reduced sites in TiO₂. A rhodium-coated SrTiO₃ powder prepared photochemically produced both H₂ and O₂ on irradiation but the turnover numbers were very low. A Rh-SrTiO₃ powder prepared thermally showed higher turnover numbers for H₂ photoproduction and may be acting catalytically. However, little O₂ was detected with this powder. When the turnover numbers for the different titanate powders were expressed with respect to the number of surface monolayer hydroxyl groups calculated from the surface area of the powders, some turnover numbers greater than one were obtained.