Purification and properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans

Abstract

The periplasmic hydrogenase of Desulfovibrio desulfuricans was isolated and purified. Cells were washed with Tris-EDTA and the enzyme precipitated from the wash with ammonium sulfate. Absorption chromatography on DEAE and hydroxyapatite yielded the enzyme at better than 95% purity as judged by gel electrophoresis. The hydrogenase catalyzed the production of more than 9000 mumol H2/min mg protein(-1) from reduced methyl viologen at 37 degrees C. It is very stable and resists inactivation by heat (50% activity remained after 5 min in air at 65 degrees C) and by enzyme inhibitors (except N-ethylmaleimide and potassium ferricyanide). After storage in air at 4 degrees C for 1 month no activity was lost. The enzyme activity is sensitive to ionic environmental changes. With methyl viologen the optimum pH was 5.5 but with p-xylene polymeric viologen the optimum was about pH 7 but less sharp. The molecular weight was 47 X 10(3)(+/- 2 X 10(3), 52 X 10(3)(+/- X 10(3), and 56 X 19(3)(+/- 2 X 10(3) by SDS-gel electrophoresis, gel chromatography, and sedimentation equilibrium, respectively, and the isoelectric point was at pH 6.0. They enzyme might be useful in the production of hydrogen from water and solar energy.

Phosphane‐ and Phosphite‐Substituted Diiron Diselenolato Complexes as Models for [FeFe]‐Hydrogenases

“…Fe 2 (µ-Se 2 C 3 H 5 CH 3 )(CO) 6 (1) was prepared according to a literature procedure. [38] Preparation of Fe 2 (µ-Se 2 C 3 H 5 CH 3 )(CO) 5 Crystal Structure Determination: The intensity data for the compounds were collected with a Nonius KappaCCD diffractometer by using graphite-monochromated Mo-K α radiation. Data were corrected for Lorentz and polarization effects, but not for absorption effects.…”

“…An important representative example of these enzymes was isolated from Desulfovibrio desulfuricans. [5,6] This enzyme can produce 9000 molecules of hydrogen per second at 30°C (hypothetically 1 mol of this enzyme could fill an airship of 13000 m 3 in about 10 min). [6] Therefore several diiron dithiolato model compounds as biomimics for the active site of this enzyme have been described (Scheme 1a).…”

“…[9][10][11][23][24][25][26][27][28][29][30] These complexes also serve as models of the active site of [FeFe]-hydrogenases. The substitution reac- [ 5 ] 2 (µ-dppe) (6). The newly synthesized complexes 2-6 were fully characterized by IR, 1 H NMR, 13 2 ] and diamines were also investigated.…”

Current Therapeutics, Their Problems, and Sulfur-Containing-Amino-Acid Metabolism as a Novel Target against Infections by “Amitochondriate” Protozoan Parasites

“…In contrast, the mostly monomeric Fe-hydrogenases usually catalyze H 2 evolution (98). In some bacteria, e.g., the hyperthermophilic bacterium Thermotoga martima and the anaerobic bacterium Desulfovibrio fructosovorans, Fe-hydrogenase is composed of two or three subunits sharing homology with monomeric Fehydrogenases (113,318). T. vaginalis possesses at least three Fe-hydrogenase genes (42,137).…”

Evidence for selenocysteine coordination to the active site nickel in the [NiFeSe]hydrogenases from Desulfovibrio baculatus.

“…The [Fe]hydrogenases contain only iron-sulfur clusters (7)(8)(9), the [NiFe]hydrogenases contain nickel and iron-sulfur clusters (10)(11)(12)(13)(14)(15), and the [NiFeSe]hydrogenases contain iron-sulfur clusters and equimolar amounts of nickel and selenium (16,17). The three types ofhydrogenase as well as multiple forms ofa single type of hydrogenase can occur within a single bacterium (18).…”