Occurrence and localization of an uptake hydrogenase in the filamentous heterocystous cyanobacteriumNostoc PCC 73102

Abstract

Free-living nitrogen-fixingNostoc PCC 73102, a filamentous heterocystous cyanobacterium originally isolated from coralloid roots of the cycadMacrozamia sp., were examined for the presence of an uptake hydrogenase (H2ase) enzyme. In vivo and in vitro hydrogen uptake measurements were used to study activities and SDS-PAGE and Western immunoblots to reveal occurrence of the hydrogenase protein. Also, transmission electron microscopy and immunocytological labeling were used to study the cellular and subcellular distribution of H2ase in theNostoc cells. In vivo measurements demonstrated an active uptake of hydrogen in both light and darkness. Light stimulated in vivo hydrogen uptake with approximately 100%, and this was further doubled by increasing the pH2, from 56 to 208 μM H2. An in vitro hydrogen uptake of 1.1 μmol H2/ mg (protein)/h was observed when using phenazinemethosulphate as e−-acceptor. Western immunoblots revealed that a polypeptide with a molecular weight of about 55 kDa was immunologically related to uptake H2ase holoenzyme purified fromAlcaligenes latus. Immunolocalization demonstrated that the H2ase protein was located both in heterocysts and vegetative cells. A higher specific labeling was associated with the cytoplasmic membranes where the vegetative cells are in contact with each other and where they actually are dividing into two vegetative cells. Using the particle analysis of an image processor, approximately equal H2ase-gold labeling per cell area was observed in the nitrogen-fixing heterocysts compared to the photosynthetic vegetative cells. This study also shows that there was no correlation between presence of phycoerythrin and uptake H2ase activity.