Properties of hydrogenase from Megasphaera elsdenii

This thesis is concerned with the purification and properties of hydrogenase from the obligate anaerobic rumen bacterium <em>Megasphaera elsdenii.</em> In chapter 1 the motives underlying this thesis, the physiological role of hydrogenase in some heterotrophs, including <em>Megasphaera elsdenii,</em> 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 <em>Megasphaera elsdenii</em> and <em>Clostridium pasteurianum</em> show great similarities.<p/>In chapter 2 the procedure for the anaerobic purification of hydrogenase from <em>Megasphaera elsdenii</em> 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 <sub><font size="-1">2</font></sub> SO and ethylene glycol (which have hydrophobic properties), and oxidants (oxygen, ferricyanide, Cl <sub><font size="-1">2</font></sub> Ind, (bi)sulphite) are described. The data show that the more chaotropic the anion of the salt the greater the increase in activity, per mole salt used. Both Me <sub><font size="-1">2</font></sub> SO and ethylene glycol inhibit the hydrogen evolution activity, however, Me <sub><font size="-1">2</font></sub> SO stimulates the hydrogen oxidation activity, while ethylene glycol does not affect this activity. Careful oxidation of the reduced enzyme with (bi)sulphite or Cl <sub><font size="-1">2</font></sub> Ind irreversibly increases the activity of the enzyme by about 60%; in contrast, oxygen and ferricyanide inactivate the enzyme. This irriversibly oxidized enzyme, which shows identical kinetic properties to the reduced enzyme, is more resistant to the effects of oxygen, Me <sub><font size="-1">2</font></sub> SO and storage, than the reduced enzyme. Difficulties encountered in ascribing redox potentials to the observed EPR spectra of hydrogenase at several redox states, such as the effects of temperature on the Nernst equation, on the apparent pH and on the midpoint potentials of the redox species, are described.<p/>In chapter 4 the theoretical aspects are described to determine the hydrogen production activity spectrophotometrically in a series coupled redox reactions as function of the pH and redox potential, as well as the limitations of this method. The hydrogen production activity is determined from the decrease in concentration of the reduced electron donor, or increase in the concentration of the oxidized electron donor. The changes in concentration of the reduced or oxidized electron donor do not equal the amount of hydrogen produced but only represent a measure of the actual amount of hydrogen produced.<p/>In chapter 5 the method as described in chapter 4 is used to study the effects of pH and redox potential on the hydrogen production activity. The hydrogen production activity is strongly pH- and redox potential-dependent; the activity patterns observed as function of pH and redox potential are independent of the nature of the electron donor used and thus represent a property of the enzyme. If at a given pH the activities are considered to express the degree of reduction of the enzyme the dependence of the activity on the redox potential represents a n=2 type redox titration curve with an 'apparent midpoint potential, which corresponds with the potential of the hydrogen electrode at that pH. To explain the effects of redox potential, proton and electron donor concentration on the hydrogenase activity Model I of chapter 2 was slightly adapted without changing its general rate equation for the hydrogen production activity. This model also explains the 'loss' of electrons of the reduced enzyme in EPR spectroscopy as well as the formation of HD together with DD during H <sub><font size="-1">2</font></sub> -D <sub><font size="-1">2</font></sub> O exchange experiments