Purification of Hydrogenase from Chlamydomonas reinhardtii

Abstract

A method is described which results in a 2750-fold purification of hydrogenase from Chiamydomone reinkardtii, yielding a preparation which is approximately 40% pure. With a saturating amount offerredoxin as the electron mediator, the specific activity of pure enzyme was calculated to be 1800 micromoles H2 produced per milligrm protein per minute. The molecular weight was determined to be 4.5 x 104 by gel filtration and 4.75 x 10i by sodium dodecyl sulfate-polyacryhlmide gel electrophoresis. The enzyme has an abundace of acidic side groups, contains iron, and has an activation energy of 55.1 kilojoules per mole for H2 production; these properties are similar to those of bacterial hydrogenases. The enzyme is less thermally stable than most bacterial hydrogenases, however, losing 50% of its activity in 1 hour at 55C. The K. of purified hydrogenase for ferredoxin is 10 micromolar, and the binding of these proteins to each other is enhanc under slightly acidic conditions. Purified hydrogenase also accepts electrons from a variety of artificial electron mediators, including sodium metatungstate, sodium silicotungstate, and several viologen dyes. A lag period is frequently observed before maximal activity is expressed with these artificial electron mediators, although the addition of sodium thiosulfate at least partially overcomes this lag.Although the biochemical properties of numerous bacterial hydrogenases have been reported, there is a distinct lack of knowledge concerning the properties of hydrogenase from eucaryotic algae. Hydrogenase has been purified from a variety of procaryotes, including Rhodospirillum (1, 2), Alcaligenes (18), Desulfovibrio (10,20), Clostridium (6), Megasphaera (21) the green alga C. reinhardtii, along with several biochemical properties of the enzyme. MATERIAILS AND METHODSCulture and Adaptation Conditions. C. reinhardtii 137C (+) was grown phototrophically under fluorescent lights ('40 w/m2) at 30°C in spinner flasks containing 12 L of minimal medium (16). Cultures were bubbled with 5% CO2 in air at a flow rate of 235 ml/min. Cells were harvested in the late exponential stage of growth (-15 gg Chl/ml) by microporous filtration (Pellicon Casette System; Millipore Corp.), washed once with 50 mm TrisCl buffer (pH 8.0) containing 3 mM MgCl2, and then resuspended in fresh buffer at a concentration of 450 to 550 ;ig Chl/ml. Cells were anaerobically adapted by bubbling Ar (99.995% purity) through the cell suspension for 4.5 h in darkened 1-L polyethylene bottles. After addition of 10 mm sodium dithionite, the bottles were tightly stoppered and stored at -20C.Assays. H2 production was measured at 25C in samples diluted into 50 mm Mops3 buffer (pH 6.8) containing 10 mM dithionite with a hydrogen electrode as described previously (16), using Fd purified from C. reinhardtii (16) as the electron mediator. One unit is defined as 1 Mmol H2 produced/min with 10 ,M Fd as the electron mediator.H2 oxidation was measured spectrophotometrically as described elsewhere (17). An extinction coefficient o...

Purification and characterization of a hydrogenase from the marine green alga Tetraselmis subcordiformis

“…Hydrogen production was investigated using different electron donors (Table 1). Methyl viologen proved to be the most efficient, as previously reported [6,8,9,18]. NAD, NADP and NADH could not be used for hydrogen production by this hydrogenase.…”

Isolation, characterization and N‐terminal amino acid sequence of hydrogenase from the green alga Chlamydomonas reinhardtii

“…Hydrogenase from C. reinhardtii was first prepared by Roessler and Lien [39]. Following their methods, we were unable to obtain homogenous preparations with high specific activities.…”

“…The enzyme was partially purified by Roessler and Lien [39]. However, their preparation could not be used for sequencing purposes.…”

Flexibility in Anaerobic Metabolism as Revealed in a Mutant of Chlamydomonas reinhardtii Lacking Hydrogenase Activity

“…The acetyl-CoA is further metabolized to ethanol and/or acetate, and the reduced ferredoxin generated by PFR1 activity is putatively oxidized by a number of redox proteins, including one or both of the two C. reinhardtii [FeFe] hydrogenases (HYDA1 and HYDA2) (16,17), which are localized to the chloroplast stroma (18,19). A recently isolated C. reinhardtii mutant, hydEF-1, is devoid of hydrogenase activity (20,21) because of disruption of an [FeFe] hydrogenase maturase, which is required for proper enzyme assembly (20 -24); consequently, neither of the hydrogenases are active in the mutant strain.…”