Properties of hydrogenase from Megasphaera elsdenii

This thesis is concerned with the purification and properties of hydrogenase from the obligate anaerobic rumen bacterium Megasphaera elsdenii. In chapter 1 the motives underlying this thesis, the physiological role of hydrogenase in some heterotrophs, including Megasphaera elsdenii, as well as a comparison of physico-chemical and kinetic properties of purified hydrogenase preparations from microorganisms, showing great variations in physiology and taxonomy, are given. The physiological role and the physico-chemical and kinetic properties of the hydrogenases from Megasphaera elsdenii and Clostridium pasteurianum show great similarities.In chapter 2 the procedure for the anaerobic purification of hydrogenase from Megasphaera elsdenii is given. Two activity bands are separated on DEAE-cellulose chromatography, of which one (fraction I; see Table 1 of this chapter) represents a different hydrogenase or a complex of the enzyme in fraction II. Kinetics with the purified enzyme of the hydrogen production activity at pH 8, with the electron donors flavodoxin hydroquinone, reduced ferredoxin and methyl viologen semiquinone show non-linear double reciprocal plots of the activity versus the electron donor concentration. Two kinetic models were developed, with an identical general rate equation for the hydrogen production activity, which describe a random mechanism for the reaction of the oxidized enzyme with a proton and a reduced electron donor. These models also indicate that the hydrogenase accepts the two electrons necessary for hydrogen production in two separate, independent steps. Hydrogen oxidation with methyl and benzyl viologen shows, in contrast to hydrogen production, Michaelis Menten kinetics. In chapter 3 effectors of the hydrogenase activity, such as salts (which have hydrophylic properties) Me 2 SO and ethylene glycol (which have hydrophobic properties), and oxidants (oxygen, ferricyanide, Cl 2 Ind, (bi)sulphite) are described. The data show that the more chaot