Inactivation of Hydrogenase in Cell-free Extracts and Whole Cells of Chlamydomonas reinhardi by Oxygen

Abstract

02 irreversibly inactivates hydrogenase from Chlamydomonas reinhardiThe mechanism for the inactivation involves the reaction of one molecule of hydrogenase with one molecule of 02 (or two oxygen atoms) in the transition complex of the rate-limiting step. The second order rate constant for this reaction is 190 atmospheres-' minute-1 (1.4 x 105 molar-' minute-1). At levels above 0.01 atmosphere 02, the increased numbers of 02 molecules may compete for the site of inactivation hindering the proper orientation for inactivation of any one 02 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 atmosphere. CO antagonizes 02 inactivation. In a period when complete inactivation by 02 would usualHy occur, the presence of CO greatly reduces the inactivation rate.After 3 hours of adaptation in whole cells, the presence of H2 lowers the rate of deadaptation of hydrogenase. Inasmuch as H2 promotes increased 02 uptake the cellular concentration of 02 is likely to be lower. After 48 hours of adaptation 02 uptake is reduced even when H2 is present and the pattern of deadaptation under 02 with and without H2 and CO is qualitatively the same as observed for the inactivation of cell-free hydrogenase. The mechanism of inactivation of celi-free hydrogenase by 02 may be the same as the mechanism for loss of hydrogenase during deadaptation in whole algal cells.Since the initial observation of H2 metabolism in green algae the sensitivity of algal H2 metabolism to O2 has been noted (5). Simultaneous uptake of H2 and O2 (the oxyhydrogen reaction) is inhibited when the O2 level exceeds a specific concentration (6, 10). Also, the photoproduction of 02 in algae will cause cessation of H2 metabolism. Removal of photoproduced O2 reversed the inhibition of hydrogenase (9) C. moewusii was grown in minimal medium plus acetate (7).The hydrogenase was prepared as above.Assays. Protein was assayed by the biuret method (8) and Chl was assayed spectrophotometrically in 80% acetone (2). The enzyme was assayed by MV3 reduction followed on a Gilford recording spectrophotometer at 605 nm. Hydrogenase solution was injected into a cuvette with 2.0 ml of anaerobically prepared 10 mM MV in 50 mm Tris-HCl (pH 8.0) under 1.0 atm H2 at 25 C. The extinction coefficient for reduced MV was determined to be 9.6 mM-' cm-' by reduction with excess sodium dithionite at pH 8.0. One unit of hydrogenase activity is defined as the reduction of 1.0 mol MV/min.

The activation of the periplasmic (NiFe) hydrogenase of Desulfovibrio gigas by carbon monoxide

“…However, no spectrophotometric evidence for the reduction of (Fe-S) centers was observed during the course of hydrogenase activation under CO. On the other hand, CO, which is a competitive inhibitor of hydrogenase activity, probably also competes with 0~ for the active centers. A similar idea has been advanced to explain the protective effect of CO against oxygen inactivation observed with a partially purified hydrogenase [15]. Argon, an inert gas with regard to metals, is ineffective in the activation of hydrogenase ( fig.2, curve 4) except for a mechanical scrubbing of oxygen which may shorten or even suppress the lag phase in the Hz-mediated activation [ 11.…”

The Role of PerR in O2-Affected Gene Expression ofClostridium acetobutylicum

“…H 2 -evolving hydrogenase A1 (HydA1) of C. acetobutylicum is highly sensitive to oxygen-dependent inactivation (11,20). While low doses of molecular O 2 lead to irreversibly damaged enzymes, CO was previously shown to act as a competitive inhibitor that antagonizes O 2 and can greatly reduce the hydrogenase inactivation state (16,43). The completely opposite expression patterns of this protein in both strains partially support this idea: the gene encoding CODH showed a high level of activation when the wild type was exposed to O 2 but the lowest level of transcription in anaerobic perR-deleted cells.…”

Purification of Hydrogenase from Chlamydomonas reinhardtii

“…In contrast, attempts to purify the enzyme from a eucaryotic source have been very limited. Erbes et aL (8) partially purified hydrogenase from C. reinhardtii in order to examine the kinetics of 02 inactivation, but the authors are unaware of any other reports concerning the purification of a eucaryotic hydrogenase.…”