The purification of hydrogenase II (uptake hydrogenase) from the anaerobic N2-fixing bacterium Clostridium pasteurianum

The H₂ uptake activity (units/mg protein) of Clostridium pasteurianum cells with methylene blue as the electron acceptor increases with cell density independent of the growth conditions. The H₂ evolution activity (units/mg protein) of the same cells with reduced methyl viologen as the electron donor remains fairly constant under all growth conditions tested. Cells grown under N₂-fixing conditions have the highest H₂ uptake activity and were used for the purification of hydrogenase II (uptake hydrogenase). Attempts to separate hydrogenase II from hydrogenase I (bidirectional hydrogenase) by a previously published method were unreliable. We report here a new large-scale purification procedure which employs a rapid membrane filtration system to fractionate cell-free extracts. Hydrogenases I and II were easily filtered into the low-molecular-weight fraction (M_r less than 100 000), and from this, hydrogenase II was further purified to a homogeneous state. Hydrogenase II is a monomeric iron-sulfur protein of molecular weight 53 000 containing eight iron atoms and eight acid-labile sulfur atoms per molecule. Hydrogenase II catalyzes both H₂ oxidation and H₂ evolution at rates of 3000 and 5.9 μmol H₂ consumed or evolved/min per mg protein, respectively. The purification procedure for hydrogenase II using the filtration system described greatly facilitates the large-scale purification of hydrogenase I and other enzymes from cell-free extracts of C. pasteurianum.     

Oxygen metabolism by the anaerobic bacteriumVeillonella alcalescens

Veillonella alcalescens contained a membrane-bound lactate oxidase system. Studies on the effect of inhibitors on lactate oxidase showed the participation of non-heme iron, quinone and cytochromesb andd. Superoxide anion radicals ( ) and H₂O₂ were shown to be formed at lactate oxidation and presumably arose from cyanide- and azide-resistant side chains of the respiratory system. The H⁺/O ratio withL-lactate as a hydrogen donor was 2.3. When an anaerobic culture growing on lactate was shifted to a high dissolved oxygen tension (d.o.t.=15 kPa) rapid inhibition of growth and lactate conversion occurred. This could be correlated with a rapid inactivation of lactate dehydrogenase. The effects of high d.o.t.'s on lactate dehydrogenase, lactate conversion and growth were reversible. After a shift to low d.o.t.'s (<2.5 kPa) growth ofV. alcalescens continued for one or two doublings whereafter lysis did occur. Acetate and pyruvate were the main fermentation products. P/O ratio's were calculated from molar growth yields and fermentation balances. A P/O value of 0.66 was found after a shift to a very low oxygen supply at which the d.o.t. presumably was zero. Shifts to higher d. o. t.'s gave much lower growth yields. Presumably, under these conditions uncoupling between growth and energy production occurred. Accumulation of toxic oxygen compounds was given as an explanation for the behaviour ofV. alcalescens at low d.o.t.'s.Abbreviations HQNO 2-n-heptyl-4-hydroxy-quinoline-N-oxide - CCCP carbonyl cyanide m-chlorophenyl-hydrazone - ABTS 2,2-azino-di-3-ethyl-benzthiazoline sulfonate - DCPIP 2,6 dichlorophenolindophenol - PMS phenazine methosulfate - NBT p-nitro blue tetrazolium chloride - d.o.t. dissolved oxygen tension - SOD superoxide dismutase     

Nitrogenase from Rhodospirillum rubrum Relation between ‘switch-off’ effect and the membrane component. Hydrogen production and acetylene reduction with different nitrogenase component ratios

Nitrogenase activity of ‘membrane-free’ extracts, produced from nitrogenstarved Rhodospirillum rubrum to which 4 mM NH⁺₄ had been added is only about 10% of the activity in the control. The activity could be restored to 80% by including the membrane component, earlier found to activate R. rubrum nitrogenase, in the reaction mixture. The relation between this ‘switch-off/switch-on’ effect and the function of the membrane component is discussed.Hydrogen production catalyzed by R. rubrum nitrogenase is also dependent on activation by the membrane component. Hydrogen production is inhibited by acetylene but the degree of inhibition is dependent on the nitrogenase component ratio. The strongest inhibition is achieved at low MoFe protein/Fe protein ratios. The $\text{ATP}\text{2 e}{}^{\mathrm{?}}$ values are 4–5 at the component ratios giving the highest activity and increase at high MoFe protein/Fe protein ratios. CO inhibits acetylene reduction but has no effect on the hydrogen production.     

Theoretical investigations on circular and chain-like hydrogen bonded structures found in two crystal forms of α-cyclodextrin hexahydrate: Models for hydration and water clusters

Hydration of macromolecules and the structure of water of crystallization are not understood in detail because in these complex systems. H-atoms cannot be located and the hydrogen bonding schemes are not known. X-ray and neutron diffraction studies on a hydrated oligosaccharide, α-cyclodextrin 6H₂O, ((C₆H₁₀O₅)₆·6H₂O), crystals forms A and B, gave insight into the chain-like and circular arrangement of hydrogen bonds. In the circles, homodromic (unidirectional) and antidromic (counter-running) orientation of five to six hydrogen bounds is observed. PCILO calculations showed that homodromic circles and chains are approx. 8% per hydrogen bond more stable than antidromic circles, that the changes in electronic charges on H and O atoms are greater in homo than in antidromic systems and that the dipole moments are only approx. 3 D in the homodromic circles but 6–8 D in chain-like and antidromic arrangement. These results have been interpreted in terms of cooperative effect. Circular systems are considered as structural elements in hydration shells of macromolecules and in the assembly of ‘flickering’ water clusters.     

Ectothiorhodospira vacuolata sp. nov., a new phototrophic bacterium from soda lakes

A new phototrophic bacterium was isolated from Jordanian and Kenyan alkaline salt lakes. Cells are rod shaped, 1.5 m wide and 2–4 m long, and motile by polar flagella. They divide by binary fission, and possess photosynthetic membranes as lamellar stacks similar to those in the other species of the genus Ectothiorhodospira and the brown colored Rhodospirillum species. The presence of bacteriochlorophyll a and carotenoids of the normal spirilloxanthin series is indicated by the absorption spectra of living cells. Under certain growth conditions the cells form gas vacuoles, may become immotile and float to the top of the culture medium. Sulfide and thiosulfate are used as photosynthetic electron donors. During the oxidation of sulfide to sulfate, elemental sulfur is formed, which is accumulated outside the cells. The organisms are strictly anaerobic, do not require vitamins, are moderately halophilic and need alkaline pH-values for growth. The new species Ectothiorhodospira vacuolata is proposed.     

Acetogenium kivui,a new thermophilic hydrogen-oxidizing acetogenic bacterium

Hydrogen-oxidizing acetogenic bacteria in pure culture are presently represented by the two mesophilic species, Acetobacterium woodii and Clostridium aceticum. From Lake Kivu we have isolated a Gram negative, chemolithotrophic, thermophilic anaerobe (LKT-1) that oxidizes hydrogen and reduces carbon dioxide to acetic acid. It is a non-motile, non-sporeforming rod, about 0.7m in width and 2–7.5m in length, often occuring in pairs or chains. The cell wall has a banded appearance; the surface layer contains a regular array of particles with six-fold rotational symmetry. No outer membrane is present. The temperature optimum for growth is 66°C, and the pH optimum is 6.4. Organic growth substrates include glucose, mannose, fructose, pyruvate, and formate; acetate is the principal product. The doubling time for growth on hydrogen and carbon dioxide is about 2h. Vitamins are neither required nor stimulatory. Yeast extract and Trypticase enhance the final yield but do not affect the growth rate. Cysteine or sulfide are required and cannot be replaced by thioglycolate or dithiothreitol. LKT-1 was mass cultured on hydrogen and carbon dioxide in a 24.1 fermentor with a yield of 34g (wet weight) of cells. The DNA base composition as determined by buoyant density is 38 mol % guanine plus cytosine. LKT-1 appears only distantly related to physiologically similar bacteria. A new genus Acetogenium is proposed, and the species is Acetogenium kivui.     

The hydrogenase-nitrogenase relationship in the blue-green algaAnabaena cylindrica

Nitrogen-fixingAnabaena cylindrica cells are found to evolve hydrogen in high quantities in the presence of CO plus C₂H₂. Studies with the inhibitors dichlorophenyldimethylurea (DCMU), disalicylidenepropanediamine (DSPD), dibromothymoquinone (DBMIB), undecylbenzimidazole (UDB) and chloro-carbonyl-cyanide-phenylhydrazone (CCCP) and also withAnabaena grown on nitrate- and ammonia-nitrogen show that the H₂-formation is due to the ATP-dependent H₃O⁺-reduction catalysed by nitrogenase. In control experiments CO plus C₂H₂ inhibited the activities of a cell-free hydrogenase fromClostridium pasteurianum. It is concluded that Anabaena has a hydrogenase whose natural function is to recycle the H₂ lost by the action of nitrogenase.Abbreviations Cl-CCP m-chloro-carbonyl-cyanide-phenylhydrazone - DSPD disalicylidenepropanediamine(1–3) - DBMIB dibromothymoquinone - DCMU N-(3,4-dichlorophenyl) NN-dimethyl-urea - UDB 2-undecyl-benzimidazole     

黄原酸酯法接枝1.丙烯酰胺和蔗渣纤维浆泊接枝共聚的研究

采用纤维素黄原酸酯——过氧化氢构成氧化还原体系,把丙烯酰胺接枝到蔗渣纤维浆泊上。探讨了引发剂用量、初始pH值、单体比、反应温度、反应时间诸因素对接枝共聚反应的影响。