Localization of the Reversible Hydrogenase in Cyanobacteria

Abstract

Abstract The reversible hydrogenase of the cyanobacterium Anacystis nidulans was purified 250-fold by classical methods. Activity staining on gels obtained by native PAGE allowed to identify two bands. A ntibodies were raised against the electrophoretically homogeneous protein. The molecu­lar weight of the hydrogenase was determined by SD S-PAGE follow ed by Western blot analysis with these antibodies. Crude extracts from the unicellular Anacystis and from heterocysts and vegetative cells of Anabaena variabilis showed precipitation bands of 56 and 17 kd. The reversible hydrogenase appeared to be com posed o f a larger and a smaller subunit as the NiFeS-hydrogen-ases from other bacteria. The hydrogenase subunits, particularly the smaller on e, were subject to proteolysis. By application of the protein A-immuno-gold labeling technique the reversible hydro­ genase was shown to associate with the cytoplasmic membrane in Anacystis. When A nacystis was grown microaerobically, the increase in the specific activity of the reversible hydrogenase paral­leled the raise in the gold-labeling at the cell periphery as measured from the electron micro­ graphs. It is concluded that Anacystis has two different hydrogenases: the reversible or bidirec­tional hydrogenase which is located exclusively at the cytoplasmic membrane of the cells, and a thylakoid-bound enzyme which catalyzes only the uptake of H2.