The organization of hydrogenase in the cytoplasmic membrane of Escherichia coli

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Abstract
The organization of the membrane-bound hydrogenase from E. coli was studied by using 2 membrane-impermeant probes, diazotized [123I]diiodosulfanilic acid and lactoperoxidase-catalyzed radioiodination. The labeling pattern of the enzyme obtained from labeled spheroplasts was compared with that from predominantly inside-out membrane vesicles, after recovery of hydrogenase by immunoprecipitation. The labeling pattern of F1-ATPase was used as a control for labeling at the cytoplasmic surface throughout these experiments. Hydrogenase (MW .apprx. 63,000) is transmembranous. Crossed immunoelectrophoresis with anti-(membrane vesicle) Ig, coupled with successive immunoadsorption of the antiserum with spheroplasts, confirmed the location of hydrogenase at the periplasmic surface. Immunoadsorption with sonicated spheroplasts suggests that the enzyme is also exposed at the cytoplasmic surface. Inside-out vesicles were prepared by agglutination of sonicated spheroplasts, and the results of immunoadsorption using these vesicles confirms the location of hydrogenase at the cytoplasmic surface.