Induction, Characterization and Localization of Cytoplasmic and Membrane Fraction Hydrogenase Enzymes in Frankia strain ArI-S
Hydrogenase enzymes in Frankia, an actinomycete found in free-living states and in symbiosis with actinorhizal plants, assist nitrogen fixation in numerous ways.l Hydrogenase activity in stirred aerobic Frankia strain ArI-5 cultures, detected using an amperometric assay sensitive to Hz concentration, was greater when incubated for five hours rather than sixteen hours in nitrogen and propionate-deficient media after brief exposure to Hz gas and growth in nitrogen-deficient media for 24-72 hours. Fresh Frankia ArI-5 cells were impermeable to Hz substrate molecules and hydrogenase activity could only be detected after freezing the cells at -20°C for at least 16 hours. Hydrogenase activity was approximately equally distributed between cytoplasmic and membrane fractions of Frankia ArI-5 separated using ultracentrifugation. Kinetic analysis of cytoplasmic and membrane hydrogenase did not yield conclusive K1/ z values. While both fractions exhibited Hz oxidation in a 200 IJ,M DTT environment, an absence of DTT yielded undetectable hydrogenase activity in the membrane fraction and H zevolution was seen in the cytoplasmic fraction. When compared to other nitrogen-fixing prokaryotes, Frankia ArI-5 membrane hydrogenase identifies with unidirectional, membrane-bound uptake hydrogenases while cytoplasmic hydrogenase resembles bidirectional, soluble hydrogenases.