Structural and catalytic properties of hydrogenase from Chromatium.
The enzyme hydrogenase, from the photosynthetic bacterium Chromatium, was purified to homogeneity after solubilization of the particulate enzyme with deoxycholate. The purification procedure included ammonium sulfate fractionation, treatment with manganous phosphate gel, heating at 63 degrees, DEAE-cellulose chromatography, and isoelectric focusing. The last step gave two active enzyme fractions with isoelectric points of 4.2 and 4.4. It was shown that the two fractions were different forms of the same protein. The enzyme was obtained in 23% yield and was purified 1700-fold. The enzyme had a molecular weight of 98,000, a sedimentation coefficient of 5.16 S and gave a single protein and activity band on disc gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis gave a single band of mol wt 50,000, suggesting that the active enzyme was composed of two subunits of the same molecular weight. The pure hydrogenase contained four atoms of iron and four atoms of acid-labile sulfide, and had a visible absorption peak at 410 nm. Electron paramagnetic resonance (EPR) spectroscopy at 10--15 K showed a free-radical signal at g' = 2.003 in the oxidized enzyme and signals at g' = 2.2 and 2.06 in the reduced enzyme. These findings suggest that Chromatium hydrogenase is an iron-sulfur protein. The pure hydrogenase catalyzed the exchange reaction between H2 and HDO or HTO, the reduction of Benzyl Viologen and Methylene Blue, and the evolution of hydrogen from reduced Methyl Viologen. The mechanism of hydrogen activation was shown to be heterolytic cleavage to an enzyme hydride and a proton. Hydrogenase could not catalyze reduction of pyridine nucleotides or ferredoxin with H2. The effect of pH and various inhibitors on the enzymatic activity has been studied.
D000596:Amino Acids / Q000032:analysis; D002844:Chromatium / Q000201:enzymology*; D002848:Chromatography, DEAE-Cellulose; D004355:Drug Stability; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000032:analysis; D007525:Isoelectric Focusing; D008970:Molecular Weight; D009243:NAD; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism; D010788:Photosynthesis; D011487:Protein Conformation; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D000596:Amino Acids; D009243:NAD; D007501:Iron; D010088:Oxidoreductases