Aerobic nitrogen fixation in Azotobacter vinelandii

cum laude graduation (with distinction)

I ELECTRON DONATION TO NITROGENASE.

Paper I shows that the hypothesis, that a high ratio of (NADH + NADPH) / (NAD + + NADP + ) is the source of reducing power for nitrogenase in intact A.vinelandii, is invalid. On the contrary, with a decreasing ratio of reduced to oxidized pyridine nucleotides, the nitrogenase activity of whole cells increases. The experiments described in paper I, indicate that the reducing power necessary for nitrogen fixation in A.vinelandii is generated within the cytoplasmic membrane. It is demonstrated that transport of reducing equivalents to the nitrogenase requires a high energy level of the cytoplasmic membrane. The energy level of the cytoplasmic membrane was measured by the intracellular Alp concentration and by using 9-amino acridine as a fluorescent probe. Other regulating factors of the nitrogenase activity in A.vinelandii are shown to be the intracellular ATP/ ADP ratio and the presence of oxygen.Paper III shows that toluene makes A.vinelandii cells permeable for small molecules but not for enzymes. In toluene-treated cells, enzyme activities can be measured by adding the appropriate cofactors and substrates. It is possible to restore the oxidation of organic substrates but no concomitant nitrogenase activity can be observed. We suggest that an observed lack of energization of the cytoplasmic membranes is the missing link between oxidation and generation of the reducing equivalents for nitrogen fixation. In paper III and IV we show that the endogenous low potential electron carriers in toluene-treated cells are not reduced. In paper IV a membrane-bound NAD(P)H-flavodoxin oxidoreductase is demonstrated and a proposal is given in which NADH is the electron donor for nitrogenase. The electron carrier flavodoxin is reduced by the membrane-bound NADH-flavodoxin oxidoreductase at a low pH, that is developed in an energy-linked process.

II OXYGEN PROTECTION OF NITROGENASE.

In paper II the source of respiration protection is investigated. Experiments with radioactive pyruvate and sucrose show that the rate of sucrose oxidation by A.vinelandii is associated with the sucrose translocator activity. We show that the respiration protection of the nitrogen-fixing system in A.vinelandii is dependent of the oxygen input during growth. The oxidation capacity intrinsically depends on the type of substrate and can be partly adapted.Membranes rich in cytochromes c 4 + c 5 and o and with phosphorylation between NADH and c 4 + c 5 and oxygen and cytochrome c 4 + c 5 and oxygen, can be isolated from A.vinelandii grown O 2 -limited. Cytochromes b and d can be detected in addition when A.vinelandii cells are grown N 2 limited. The activity of the NADH oxidase system is increased in such cells and phosphorylation is only observed between CoQ and oxygen. Under saturating oxygen concentrations the type of respiratory membranes was not observed to influence the intracellular energy charge.In paper III and IV the mechanism of the conformational protection of nitrogenase was investigated. It is shown that nitrogenase can be isolated as an oxygen-stable complex form A.vinelandii independent of the cell rupture method. Also no influence of the cell rupture method on the rate of sedimentation of the nitrogenase can be observed. The rate of sedimentation of the nitrogenase is found to be concentration and pH dependent. At pH=7.4 the rate of sedimentation of the nitrogenase complex is comparable with that of the pyruvate dehydrogenase complex.No evidence was found for a particulate nitrogenase, it is demonstrated that the oxygen stability of nitrogenase in crude extracts is caused by complexation. of the nitrogenase components with an Fe-S protein. An alternative proposal for the switch-on switch-off phenomenon in whole Azotobacter cells is given. Nitrogenase is present in vivo as an active and oxygen tolerent complex but nitrogen fixation in whole cells is inhibited by the oxidation of flavodoxin hydroquinone.