Cees Veeger

Separation of hydrogenase from intact cells of Desulfovibrio vulgaris Purification and properties

(Hildenborough [l-3] and Miyazaki [4,5] ). In most of the purification procedures that have been described the bacteria were broken mechanically, and in the case of the Miyazaki strain, a subsequent treatment with pancreatin or trypsin was found to be required to solubilize the enzyme [4,5]. The recoveries of activity after purification were low (0.4-2.8%), and purified preparations from a single strain differed in a number of properties, including molecular weight, subunit composition and iron content [2,3]. Part of the hydrogenase activity in a related organism,

Enzymatic conversion of apolar compounds in organic media using an NADH regenerating system and dihydrogen as reductant

A combined enzyme system, consisting of hydrogenase, lipoamide dehydrogenase and 20β‐hydroxysteroid dehydrogenase has been enclosed in reversed micelles. This system catalyzes the stereo‐ and site‐specific enzymatic reduction of apolar, poorly water‐soluble ketosteroids to their corresponding 20β‐hydroxyform using an in situ NADH‐regenerating enzyme system and H2 as ultimate reductant.

[16] Succinate dehydrogenase: [EC 1.3.99.1 Succinate: (acceptor) oxidoreductase]

Publisher Summary The chapter describes two methods for the determination of the activity of soluble succinate dehydrogenase with artificial electrons acceptors. The first method, a manometric one with Phenazine Methyl Sulfate (PMS), is modified for spectrophotometric use. The rates obtained with the spectrophotometric modification are not proportional with the enzyme concentration. However, when a suitable control lacking succinate is used, the rates are closely proportional. The second method, which uses K3Fe(CN)6 as acceptor, is more convenient as a routine assay. The method of determination of the reconstitution activity of a soluble, purified succinate dehydrogenase preparation is also described. The purification procedure described comprises of 2 parts: preparation of cytochrome c-deficient heart muscle preparation; preparation of the soluble enzyme. The enzyme is very unstable at room temperature even when kept under N2. Under all conditions, the reconstitution activity declines faster than the activity with PMS and K3Fe(CN)6. The reconstitution activity is fairly stable on storage under liquid N∼; the activity with hydrogen acceptors is also stable under these conditions.

Electrochemical behaviour of low-potential electron-transferring proteins at the mercury electrode

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MOLECULAR LUMINESCENCE OF SOME ISOALLOXAZINES IN APOLAR SOLVENTS AT VARIOUS TEMPERATURES

Abstract— –Spectral properties of isoalloxazines in organic solvents of low polarity are determined at 300 and 77 K. Vibrational structure in the spectra reveals a vibrational mode of 1250cm‐. The pure electronic transition energies are established to a greater accuracy than was done previously and comparison to theoretical data is made. Actual lifetimes up to 10 ns for fluorescence and 300 ms for phosphorescence are found. The ratio of the actual fluorescence lifetime and the radiative lifetime is found to agree well with the quantum yield. Solvent interactions hardly shift the energy of the first electronically excited singlet state but merely affect the Franck‐Condon envelope of the spectrum and the non radiative decay of the chromophore. In albne solutions at 77 K isoalloxazine clusters are formed exhibiting P‐type delayed fluorescence.

Regulation of dinitrogen fixation in intact Azotobacter vinelandii

Abstract 1. In intact Azotobacter vinelandii the influence of oxygen on the levels of oxidized nicotinamide adenine dinucleotides and adenine nucleotides in relation to nitrogenase activity was investigated. 2. The hypothesis that a high ( NADH + NADPH ) ( NAD + + NADP + ) is the driving force for the transport of reducing equivalents to nitrogenase in intact A. vinelandii was found to be invalid. On the contrary, with a decreasing ratio of reduced to oxidized pyridine nucleotides, the nitrogenase activity of the whole cells increases. 3. By measuring oxidative phosphorylation and using 9-amino acridine as a fluorescent probe, it could be demonstrated that respiration-coupled transport of reducing equivalents to the nitrogenase requires a high energy level of the plasma membrane or possibly coupled to it, a high pH gradient over the cytoplasmic membrane. Furthermore nitrogen fixation is controlled by the presence of oxygen and the ATP/ADP ratio.

The effect of re-oxidation on the reduced hydrogenase of Desulfovibrio vulgaris strain Hildenborough and its oxygen stability

Upon reduction of the oxygen‐stable hydrogenase of Desulfovibrio vulgaris strain Hildenborough this enzyme becomes oxygen sensitive. Only anaerobic oxidation of the reduced enzyme with 2,6‐dichlorophenol‐indophenol, in the presence of iron and EDTA, followed by addition of Tris‐EDTA (pH 9.0) converts the enzyme back into its oxygen‐stable state, without loss of activity.

Does P450-type catalysis proceed through a peroxo-iron intermediate? A review of studies with microperoxidase

Recent stopped-flow kinetics demonstrated the existence of an intermediate before the occurrence of the final product of the reaction of both iron-containing microperoxidase-8 (Fe(III)MP-8) and manganese-containing microperoxidase-8 (Mn(III)MP-8) with H(2)O(2). The intermediate was assigned to be (hydro)peroxo-iron. With both mini-catalysts the final state obtained after 30-40 ms showed a resemblance to PorM(IV)MP-8[double bond]O(R(+)*); (R(+)*) is a radical located at the peptide. Quantum mechanical calculations indicate that hydroperoxo-iron is inactive as a catalytic intermediate in cytochrome P450 (P450)-type catalysis. Instead, the calculations suggest that peroxo-iron acts as the catalytic intermediate in P450-type catalysis. In addition, the calculations demonstrate that, although less likely, the possibility that oxenoid-iron acts as a catalytic intermediate in P450 catalysis cannot be fully excluded. An interesting aspect of the reactions catalysed by MP-8 is the possibility that, in view of the reversibility of the reactions between (hydro)peroxo-iron and oxenoid-iron, H(2)O plays a decisive role, at least in some cytochromes P450, in the removal of halogens, avoiding the production of compounds hazardous to the organism.

Design of reversed micellar media for the enzymatic synthesis of apolar compounds

Publisher Summary This chapter focuses on the reversed micellar enzymology of 20 β-hydroxysteroid dehydrogenase that catalyzes the reduction of various ketosteroids to the corresponding 20 β-hydroxysteroids at the expense of NADH. Reversed micelles are tiny oases of water stabilized in an organic solvent with the aid of surfactants. It shows that enzymes entrapped in these pools are capable of synthesizing apolar compounds—such as steroids, oxidized polyunsaturated fatty acids, and peptides. A reversed micellar medium consists of at least three components: an aqueous solution, a water-immiscible organic solvent, and a surfactant. The mechanism leading to the formation of reversed micelles is the tendency to extend the interfacial area until the concentrations of surfactants are sufficiently low to achieve a nonnegative interfacial tension. The results demonstrate that the reaction rate declines when the conversion is almost complete. Furthermore, at higher concentrations the reaction rate increases and the reaction proceeds steadily for >10 hr, indicating that the multienzyme system is stable for a considerable length of time.

The involvement of the membrane potential in nitrogen fixation by bacteroids of Rhizobium leguminosarum

Department of Biochemist~T, Agricultural Unh~ersity, 6703 BC Wagenb~gen and *Department of 11~icrobiology. Ri]ksunh,ersiteit Groni~gen, 9751 NIV Haren, The Netherlands Received 2! May 1979 1. Introduction One of the major problems concerning nitrogen fixation in obligate aerobes is the generation of reduc- ing equivalents for nitrogenase. Current evidence witb