Electron paramagnetic resonance and other properties of hydrogenases isolated from D. vulgaris (strain Hildenborough) and M. elsdenii (original) (raw)
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European Journal of Biochemistry, 1983
The hydrogenases of DesuEfovibrio vulgaris and Megasphaera elsdenii are compared with respect to some of their physical properties. In addition to Fe the only metal ions that are present in significant amounts are Ni and Cu. From cluster extrusion experiments it follows that the D. vulgaris enzyme contains three 4 Fe-4 S clusters, while M . elsdenii hydrogenase only releases part of its Fe-S clusters. The resting D . vulgarisenzyme shows only a small 3 Fe-xS type of EPR signal (maximum 5 %electronequivalent). This amount can beincreased to approximately25 %by treatment with ferricyanide, with a concomitant large decrease in activity. The M. elsdenii enzyme shows in its oxidized state a normal Hipip (high-potential iron-sulphur protein) type of EPR spectrum. After a reduction/oxidation cycle the D. vulgaris enzyme also shows a weak Hipip type of EPR spectrum. In the reduced state both enzymes show complex spectra. By integration of those spectra it is shown that 1.5 electron equivalents are present. The complex spectra do not arise from nuclear hyperfine interactions but are partially due to electron spin interactions. It is proposed that the spectrum of reduced D. vulgaris hydrogenase consists of a sum of three different ferredoxin-like spectra.
Properties of the Hydrogenase of Megasphaera elsdenii
European Journal of Biochemistry, 1980
The catalytic activities of Megusplzueru elsdenii hydrogenase are stimulated by salts. The stimulation is due to the anion: the more cliaotropic the anion, the greater the effect.
JBIC Journal of Biological Inorganic Chemistry, 2002
Hydrons and electrons are substrates for the enzyme hydrogenase, but cannot be observed in X-ray crystal structures. High-resolution 1 H electron nuclear double resonance (ENDOR) spectroscopy oers a means to detect the distribution of protons and unpaired electrons. ENDOR spectra were recorded from frozen solutions of the nickel-iron hydrogenases of Desulfovibrio gigas and Desulfomicrobium baculatum, in thè`a ctive'' state (``Ni-C'' EPR signal) and analyzed by orientationally selective simulation methods. The experimental spectra were ®tted using a structural model of the nickel-iron centre based on crystallographic results, allowing for dierences in electron spin distribution as well as the spatial orientation of the g-matrix (g-tensor), and anisotropic and isotropic hyper®ne couplings of the protons nearest to the nickel ion. ENDOR signals, detected after complete deuterium exchange, were assigned to six protons of the cysteines bound to nickel. The assignment took advantage of the substitution of a selenium for a sulfur ligand, which occurs naturally between the [NiFeSe] and [NiFe] hydrogenases from Dm. baculatum and D. gigas, respectively, and was found to aect just two signals. The four signals with the largest hyper®ne couplings, including isotropic contributions from 4.5 to 13.5 MHz, were assigned to the bmethylene protons of the two terminal cysteine ligands, one of which is substituted by seleno-cysteine in [NiFeSe] hydrogenase. The electron spin is delocalized onto the nickel (50%) and its sulfur ligands, with a higher proportion on the terminal than the bridging ligands. The g-matrix was found to align with the active site in such a way that the g 1 -g 2 plane is nearly coplanar (18.3°) with the plane de®ned by nickel and three sulfur atoms, and the g 2 axis deviates by 22.9°from the vector between nickel and iron. Signi®cantly for the reaction of the enzyme, direct evidence for the binding of hydrons at the active site was obtained by the detection of H/Dexchangeable ENDOR signals.
Purification and Properties of Hydrogenase from Megasphaera elsdenii
European Journal of Biochemistry, 1979
A hydrogenase has been purified to homogeneity from the soluble fraction of the rumen bacterium Megasphaera elsdenii, the overall purification is 200 times with a yield of 14%. The pure enzyme consists of a single polypeptide chain with M , % 50000 which contains 12 atoms of non-haem iron and 12 atoms of acid-labile sulphide. The enzyme is rapidly inactivated by 0 2 and it is therefore purified under nitrogen and in the presence of sodium dithionite. The optical spectrum of the enzyme, after removal of the dithionite with air, shows a peak at 275 nm (8275 nm = 143 mM-' cm-') and a shoulder between 350 nm and 400 nm (w0 , , , , , = 46 mM-' cm-I).
Fe-only hydrogenases: structure, function and evolution
Journal of Inorganic Biochemistry, 2002
Hydrogenases are enzymes capable of catalyzing the oxidation of molecular hydrogen or its production from protons and electrons 1 2 according to the reversible reaction: H á2H 12e . Most of these enzymes fall into to major classes: NiFe and Fe-only hydrogenases.