Abstract
Two distinct processes are involved in the formation of active hydrogenase during anaerobic adaptation of Chlamydomonas reinhardtii cells. In the first 30 minutes of anaerobiosis, nearly all of the hydrogenase activity can be attributed to activation of a constituitive polypeptide precursor, based on the insensitivity of the process to treatment with cycloheximide (15 micrograms per milliliter). This concentration of cycloheximide inhibits protein synthesis by greater than 98%. After the initial activation period, de novo protein synthesis plays a critical role in the adaptation process since cycloheximide inhibits the expression of hydrogense in maximally adapted cells by 70%. Chloramphenicol (500 micrograms per milliliter) has a much lesser effect on the adaptation process.Incubation of cell-free extracts under anaerobic conditions in the presence of dithionite, dithiothreitol, NADH, NADP, ferredoxin, ATP, Mg(2+), Ca(2+), and iron does not lead to active hydrogenase formation. Futhermore, in vivo reactivation of oxygen-inactivated hydrogenase does not appear to take place.The adaptation process is very sensitive to the availability of iron. Iron-deficient cultures lose the ability to form active hydrogenase before growth, photosynthesis, and respiration are significantly affected. Preincubation of iron-deficient cells with iron 2 hours prior to the adaptation period fully restores the capacity of the cells to synthesize functional hydrogenase.