Purification of Hydrogenase from Chlamydomonas reinhardtii.

A method is described which results in a 2750-fold purification of hydrogenase from Chlamydomonas reinhardtii, yielding a preparation which is approximately 40% pure. With a saturating amount of ferredoxin as the electron mediator, the specific activity of pure enzyme was calculated to be 1800 micromoles H(2) produced per milligram protein per minute. The molecular weight was determined to be 4.5 x 10(4) by gel filtration and 4.75 x 10(4) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme has an abundance of acidic side groups, contains iron, and has an activation energy of 55.1 kilojoules per mole for H(2) 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 55 degrees C. The K(m) of purified hydrogenase for ferredoxin is 10 micromolar, and the binding of these proteins to each other is enhanced 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.