INACTIVATION OF HYDROGENASE IN CELL-FREE-EXTRACTS AND WHOLE CELLS OF CHLAMYDOMONAS-REINHARDI BY OXYGEN

O2 irreversibly inactivates hydrogenase from C. reinhardi. The mechanism for the inactivation involves the reaction of 1 molecule of hydrogenase with 1 molecule of O2 (or 2 O atoms) in the transition complex of the rate-limiting step. The second order rate constant for this reaction is 190 atm-1 min-1 (1.4 .times. 105 M-1 min-1). At levels above 0.01 atm O2, the increased numbers of O2 molecules may compete for the site of inactivation hindering the proper orientation for inactivation of any one O2 molecule and resulting in lowered rates of inactivation. CO is a reversible inhibitor of hydrogenase acting competitively against H2. The Ki for CO is 0.0010 atm. CO antagonizes O2 inactivation. In a period when complete inactivation by O2 would usually occur, the presence of CO greatly reduces the inactivation rate. After 3 h of adaptation in whole cells, the presence of H2 lowers the rate of deadaptation of hydrogenase. Inasmuch as H2 promotes increased O2 uptake, the cellular concentration of O2 is likely to be lower. After 48 h of adaptation O2 uptake is reduced even when H2 is present and the pattern of deadaptation under O2 with and without H2 and CO is qualitatively the same as observed for the inactivation of cell-free hydrogenase. The mechanism of inactivation of cell-free hydrogenase by O2 may be the same as the mechanism for loss of hydrogenase during deadaptation in whole algal cells.