Solar energy conversion by green microalgae: a photosystem for hydrogen peroxide production.

A photosystem for solar energy conversion, comprised of a culture of green microalgae supplemented with methyl viologen, is proposed. The capture of solar energy is based on the Mehler reaction. The reduction of methyl viologen by the photosynthetic apparatus and its subsequent reoxidation by oxygen produces hydrogen peroxide. This is a rich-energy compound that can be used as a nonpollutant and efficient fuel. Four different species of green microalgae, Chlamydomonas reinhardtii (21gr) C. reinhardtii (CW15), Chlorella fusca, and Monoraphidium braunii, were tested as a possible biocatalyst. Each species presented a different efficiency level in the transformation of energy. Azide was an efficient inhibitor of the hydrogen peroxide scavenging system while maintaining photosynthetic activity of the microalgae, and thus significantly increasing the production of the photosystem. The strain C. reinhardtii (21gr), among the species studied, was the most efficient with an initial production rate of 185 micromol H(2)O(2)/h x mg Chl and reaching a maximum of 42.5 micromol H(2)O(2)/mg Chl when assayed in the presence of azide inhibitor.

A flow injection chemiluminescence method using Cr(III) as a catalyst for determining hydrogen peroxide. Application to H(2)O(2) determination in cultures of microalgae.

Flow injection analysis has been applied to the determination of hydrogen peroxide produced by some different species of microalgae. The method is based on the luminol-H(2)O(2) chemiluminescence reaction using Cr(III) as a catalyst. Optimum experimental conditions for the method have been studied and trace amounts of hydrogen peroxide determined with detection limits of 4 10(-8) mol/L. The method using Cr(III) was compared with that using horseradish peroxidase as the catalyst.

Laser flash photolysis studies of electron transfer in complex III from yeast mitochondria.

The kinetics of reduction of cytochrome b and cytochrome c1 of yeast Complex III by 5-deazariboflavin semiquinone, generated by laser flash photolysis under anaerobic conditions, have been investigated. The reduction of cytochrome b occurs in two phases with first-order rate constants of 1300 and 670 s-1, whereas the reduction of cytochrome c1 appears as a unique exponential phase with an intermediate value of 800 s-1. Under these experimental conditions, about 50% of cytochrome b is reduced in comparison with cytochrome c1. After photoreduction, the re-oxidation of the cytochromes by internal re-equilibrium occurs in both cases, following pseudo-first-order kinetics at a rate constant of 43 s-1 for cytochrome b and 39 s-1 for cytochrome c1. These results, which agree with the data from the rapid mixing technique (A.-L. Tsai, J.S. Olson, G. Palmer, J. Biol. Chem. 262 (1987) 8677-8684), have implications for the mechanistic understanding of inner Complex III electron transfer. One of the goals of the investigation reported here is to provide direct evidence for the hypothesis of a proton-motive Q cycle for the mechanism of electron transfer in Complex III. Moreover, these results demonstrate the usefulness of laser flash photolysis in studying the redox kinetic properties of mitochondrial Complex III.

Storage of solar energy by production of hydrogen peroxide by the blue-green alga Anacystis nidulans R2: stimulation by azide.

The production of hydrogen peroxide by Anacystis nidulans R2 in presence of methyl viologen occurs by using the redox power from water promoted by the photosystems of the blue-green alga. Thus, in the presence of the photosynthetic inhibitor DCMU or in the dark, H(2)O(2) production does not take place. In cells permeabilized with lysozyme, the addition of ionophores, which is expected to increase the electron flow, produces only a small increase to initial velocity of hydrogen peroxide production. On the other hand, in nonpermeabilized cells, the addition of superoxide dismutase increases the initial velocity of hydrogen peroxide production, but the net amount accumulated by the system is very low because of posterior decomposition. Preincubation of cells with azide, which inhibits the catalase, prevents the decomposition, thereby increasing drastically the amount of hydrogen peroxide accumulated by the system after a few hours. Hence, H(2)O(2) production appears to be limited mainly because of decomposition by catalase activity rather than by the photosynthetic electron flow rate or the diffusion of products through the cell wall. The net production of hydrogen peroxide by the system was enhanced severalfold by treatment with azide. If one takes into account the use of hydrogen peroxide as fuel due to the large amount of energy released in its dismutation, the photosystem can be a useful tool in the storage of solar energy.

Restoration of high-potential cytochrome b-564 by integration of baker's yeast complex III into liposomes.

Cytochrome b-564 in isolated complex III from baker's yeast mitochondria exhibits the midpoint redox potential proper to the low-potential couple (+60 mV, pH 7.2). Incorporation of the complex into liposomes promotes total conversion to the high-potential couple (+170 mV, pH 7.2). The reconstituted system shows electrogenic proton translocation, which is inhibited by the uncoupler CCCP. Deenergizing treatments result, moreover, in reversal of the redox potential change. These results support our previous proposal that cytochrome b-564 acts as a transducer of redox energy into acid-base energy in the complex III region of the respiratory chain.

Coupling between redox and acid-base energy by cytochrome b-564 in Baker's yeast mitochondria.

Baker's yeast mitochondrial cytochrome b-564 is characterized by exhibiting both a labile pH-independent high-potential form (E'o, pH 7 = + 190 mV) and a stable pH-dependent (pKa = 6.8) low-potential form (E'o, pH 7 = + 70 mV). The different behavior of these two forms of cytochrome b-564 versus pH seems to be a decisive factor for transduction of redox energy into acid-base energy in oxidative phosphorylation site 2. Deenergizing treatments, such as ADP plus Pi, result in the conversion of all the mitochondrial cytochrome b-564 into its low-potential form, whereas energization with ATP specifically transforms the cytochrome into its high-potential form, the ATP effect being neutralized by the ATPase inhibitor oligomycin and by the uncoupler FCCP. Accordingly, a minimal model for coupling between redox energy and acid-base energy through an electronically energized and protonated ferricytochrome b-564 intermediate is proposed. The energy-transducing properties of mitochondrial cytochrome b-564 seems to be shared by chloroplast cytochrome b-559.

Anomalous reduction of cytochrome b in highly purified complex III from baker's yeast.

In highly purified bc1-complex from baker's yeast, the reduction of cyt c1 and partial reduction of cyt b is obtained by catalytic amount of succinate dehydrogenase and succinate in the presence of 7 microM antimycin. After the addition of ferricyanide the c1 is re-oxidized and a increase in the reduction of b is observed. Using stopped-flow we established that the oxidation of c1 by ferricyanide proceeds as a pseudo-first order reaction and the reduction of b is faster and with two phases. Our observation suggests that these two processes are not directly interconnected and that other component than c1 must be the "control factor" in the anomalous reduction of cyt b. This component must be, by exclusion, the iron-sulfur protein.

Studies on the kinetic mechanism of nitrate reductase from spinach (Spinacea oleracea).

Based on Lineweaver-Burk plots of the initial velocities, at different concentrations of NADH and nitrate, and product inhibition patterns, an Iso Ping Pong Bi Bi steady state kinetic mechanism is proposed for the spinach nitrate reductase. This mechanism incorporates the concept that the oxidized enzyme is present in two isomeric forms.

The preparation and characterization of highly purified, enzymically active complex III from baker's yeast.

A soluble enzymically active cytochrome b.c1 complex has been purified from baker's yeast mitochondria by a procedure involving solubilization in cholate, differential fractionation with ammonium sulfate, and ultracentrifugation. The resulting particle is free of both cytochrome c oxidase and succinate dehydrogenase activities. The complex contains cytochromes b and c1 in a ratio of 2:1 and quinone and iron-sulfur protein in amounts roughly stoichiometric with cytochrome c1. EPR spectroscopy has shown the iron-sulfur protein to be present mainly as the Rieske protein. EPR spectroscopy also shows a heterogeneity in the cytochrome b population with resonances appearing at g = 3.60 (cytochrome bK) and g = 3.76 (cytochrome bT). A third EPR resonance appearing in the region associated with low spin ferric hemes (g = 3.49) is assigned to cytochrome c1. Anaerobic titration of the complex with dithionite confirmed the heterogeneity in the cytochrome b population and demonstrated that the oxidation-reduction potential of the iron-sulfur protein is approximately 30 mV more positive than cytochrome c1. An intense EPR signal assigned to the coenzyme Q free radical appeared midway in the reductive titration; this signal disappeared toward the end of the titration. A conformational change in the iron-sulfur protein attendant on reduction of a low potential species was noted.

Nitrate reductase from Spinacea oleracea. FAD and the reactivation of the enzyme treated with p-Hydroxymercuribenzoate.

Spinach nitrate reductase complex previously inactivated by treatment with mercurials p-hydroxymercuribenzoate or p-hydroxymercuriphenyl sulphonate can be reactivated by incubation with dithioerythritol. The reactivation of NADH-diaphorase seems to be FAD-dependent, whereas that of FNH2-nitrate reductase is not. The requirement of FAD for NADH-inactivation of nitrate reductase treated with p-hydroxymercuribenzoate disappears after treatment with dithioerythritol.