Alvin I. Krasna

PROFLAVIN CATALYZED PHOTOPRODUCTION OF HYDROGEN FROM ORGANIC COMPOUNDS

Abstract— Methyl viologen was reduced by EDTA and other organic compounds when aqueous solutions of these compounds were irradiated near 440 nm in the presence of catalytic quantities of proflavin. The photoreduced methyl viologen was readily oxidized in the dark by the enzyme hydrogenase or platinum asbestos. When the entire reaction was run in the light with hydrogenase or platinum, continuous production of hydrogen was observed. The yield of hydrogen was approximately stoichiometric to the EDTA present establishing that methyl viologen and proflavin were acting catalytically. To establish the structural requirements of the electron donor, eighty compounds were tested at seven pH values between 4 and 10. Of these, twenty served as electron donors for the photoproduction of hydrogen. The effective donors contained either a secondary or tertiary nitrogen atom with one or more carboxymethyl or β‐hydroxyethyl groups, or a sulfhydryl group. The system could also reduce benzyl viologen but not methylene blue. Riboflavin did not replace proflavin for the photoproduction of hydrogen, This system may have potential for producing hydrogen with solar energy.

Hydrogenase: Properties and applications

Abstract Hydrogenase is an enzyme which reversibly activates molecular hydrogen and has potential applications in the production of hydrogen by solar energy. This review describes methods employed for assay of the enzyme, the biological role of hydrogenase in normal cellular metabolism and growth, and the properties of the enzyme. Although hydrogenases isolated from different organisms differ in molecular weight, subunit composition, iron and sulphur content, thermal and oxygen stability, they are all iron-sulphur proteins which cleave hydrogen heterolytically to form a hydride and a proton. The review also describes various systems being developed for the biophotolysis of water to produce hydrogen, and the role of hydrogenase in these systems.

Photoproduction of hydrogen from water in hydrogenase-containing algae

Abstract Scenedesmus obliquus and Chlorella vulgaris cells had active hydrogenase after dark anaerobic adaptation. Illumination of these algae with visible light led to an initial production of small quantities of hydrogen gas which soon ceased owing to production of oxygen by photolysis of water. The presence of oxygen-absorbing systems in a separate chamber, not in contact with the algae, gave only a slight stimulation of hydrogen production. Addition of sodium dithionite directly to the algae led to an extensive light-dependent production of hydrogen. This stimulation was due to oxygen removal by dithionite and not to its serving as an electron donor. 3-(3,4-Dichlorophenyl)-1,1-dimethylurea, an inhibitor of photosystem II, abolished all hydrogen photoproduction. Hydrogen evolution was not accompanied by CO2 production and little difference was noted between autotrophically and heterotrophically grown cells. Hydrogen was not produced in a photosystem II mutant of Scenedesmus even in the presence of dithionite, establishing that water was the source of hydrogen via photosystems II and I. Hydrogen production was stimulated by the presence of glucose and glucose oxidase as an oxygen-absorbing system. Oxygen inhibited hydrogen photoproduction, even if oxygen was undetectable in the gas phase, if the algal solution did not contain an oxygen absorber. It was demonstrated that under these conditions hydrogenase was still active and the inability to produce hydrogen was probably due to oxidation of the coupling electron carrier.

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.

Solubilization and properties of the hydrogenase of Chromatium

Abstract Cell-free extracts of Chromatium were separated into a soluble and a particulate chromatophore fraction and the latter fraction contained most of the hydrogenase activity of the cells as well as the ferrodoxin-dependent H 2 NAD + reducing activity. The particulate hydrogenase was solubilized by treating either cells or particles with Triton X-100 or deoxycholate. The solubilized hydrogenase prepared from cells could reduce NAD + in the presence of ferrodoxin while the solubilized hydrogenase prepared from particles could not. Benzyl viologen stimulated NAD + reduction in all preparations. The hydrogenase of Chromatium activates H 2 by a heterolytic split to form an enzyme hydride.

LIMITING REACTIONS IN HYDROGEN PHOTOPRODUCTION BY CHLOROPLASTS AND HYDROGENASE

Abstract— Hydrogen was photoproduced from water in a system containing isolated chloroplasts, hy‐drogenase, a coupling electron carrier (ferredoxin or methyl viologen), and an oxygen scavenger. The rate and extent of hydrogen production anaerobically was much less than the rate of aerobic electron‐carrier reduction by chloroplasts and was not limited by hydrogenase. The limiting reaction in the coupled system was the extent of reduction of methyl viologen anaerobically rather than its oxidation by oxygen produced during the course of the reaction. Inhibition of photosystem II by 3‐(3,4dichlorophenyl)‐1,1‐dimethylurea and addition of a photosystem 1 electron donor did not lead to photoproduction of hydrogen or photoreduction of methyl viologen. Extensive photosystem I hydrogen evolution was obtained when thiols were also present. Platinum asbestos or palladium asbestos replaced hydrogenase in a system coupled to chloroplasts.

Characterization and stability of hydrogenase from Chromatium

The absorption spectrum of the hydrogenase from Chromatium, which contains four iron atoms and four atoms of acid-labile sulfide, in 80% dimethylsulfoxide or hexamethylphosphoramide suggests the presence of a single [4Fe-4S] cluster. The EPR spectra of the oxidized enzyme in air, argon or carbon monoxide are the same with signals centered at g = 2.01. The enzyme reduced by hydrogen is EPR silent. The EPR spectrum is consistent with a [4Fe-4S] cluster. Chromatium hydrogenase and the hydrogenase from Proteus vulgaris show relative stability towards denaturation by sodium dodecyl sulfate (SDS), urea, guanidine and organic solvents.

Properties of the hydrogenase of scenedesmus

1. 1. The adaptation of hydrogenase (EC 1.98.1.1) in Scenedesnus obliquus was inhibited by dithionite, sulfite, arsenite, marphaside, BAL, 2,2′-dipyridyl, and 1,10-phenanthroline. None of these inhibitors had any effect on the active hydrogenase after adaptation. 2. 2. The active hydrogenase of Scenedesmus was inhibited by silver, mercury, and cupric ions, p-chloromercuribenzoate and iodoacetamide. The silver and mercury inhibition could be reversed by BAL. 3. 3. The hydrogenase of Proteus vulgaris was not affected by the inhibitors of the adaptation process but was inhibited by those reagents which inhibited the active hydrogenase of Scenedesmus.

Purification and properties of the light-activated hydrogenase of Proteus vulgaris

Abstract The light activation of the hydrogenase of Proteus vulgaris previuosly observed in whole cells has been demonstrated in cell-free extracts and in the particulate hydrogenase obtained from such extracts. The particulate hydrogenase was solubilized by treating it with deoxycholate at pH 8.0 and was further purified by (NH 4 ) 2 SO 4 fractionation, by heating at 60° and by Sephadex chromatography. This procedure afforded a 65-fold purification, and all fractions exhibited light activation of the hydrogenase, suggesting that light directly affects the hydrogenase. A comparison of the light-activated purified enzyme and the one active in the dark showed that there was no difference in the molecular weight (115 000), in the mechanism of hydrogen activation or in the K m for hydrogen, while v max was greater for the light-activated enzyme. The mechanism of light activation is discussed in terms of these findings.

Changes in the light-scattering properties of DNA on denaturation

Abstract Light scattering measurements made at angles above 25° do not give the correct molecular weight for native DNA. By construction of a low-angle instrument and using new methods to clarify the sample, measurements could be made down to 10°. The low-angle data (10–25°) for calf-thymus and T7 DNA gave a molecular weight twice that obtained at high angles (26–60°). The low-angle molecular weights agreed with those obtained from sedimentation and viscosity measurements. Denaturation of DNA by acid, alkali, or heat caused a halving of the molecular weight, a large decrease in the radius of gyration, and a loss of viscosity. This result was only obtained with low-angle measurements; high-angle measurements showed no decrease in molecular weight on denaturation. On lowering the Na + concentration progressively from 10 −1 to 10 −5 M , there was a linear decrease in the T m and an increase in the intrinsic viscosity. Low-angle light scattering showed that at 10 −3 M salt the molecular weight was one-fourth that observed at 10 −1 or 10 −2 M salt, while at 10 −1 and 10 −2 M salt the solutions did not scatter light at all. When the salt concentration of the latter solutions was raised to 10 −1 M , scattering took place and the molecular weight was that expected for denatured DNA. Identical results were obtained when heat-denatured DNA was studied in low salt suggesting that the properties observed are due to the polyelectrolyte effect in single-stranded DNA and not to abnormal denaturation.