Purification and properties of hydrogenase from Clostridium pasteurianum W5

Abstract

Hydrogenase from Clostridium pasteurianum W5 prepared by a previously reported method was found to be heterogeneous. A new procedure has been developed which achieved a 320-fold purification with an 11% recovery of the enzyme activity. This represents a 6.6-fold increase in specific activity over the previously reported value. Such preparations showed a maximal activity ( V) of about 4000 μmoles H 2 evolved/min/mg protein when assayed in the presence of ferredoxin and 1 mM methyl viologen. Hydrogenase so prepared has a molecular weight of 60 500 and did not dissociate into subunits when treated with sodium dodecylsulfate and 2-mercaptoethanol. Hydrogenase has 12 half-cystine residues per molecule and a preponderance of acidic amino acids. This hydrogenase does not contain molybdenum or copper but has about 12 iron atoms and 12 acid-labile sulfur groups per molecule. All of the iron atoms are resistant to anaerobic treatment with o- phenanthroline . Methyl viologen, benzyl viologen, methylene blue and ferredoxin can each serve as the sole electron carrier for the purified enzyme.