Purification and some properties of the soluble hydrogenase from Chromatium vinosum.
A routine procedure for the growth and harvesting of large (600 1) batches of Chromatium vinosum and the isolation of hydrogenase (hydrogen: (acceptor) oxidoreductase, EC 1.12.-.-) are described. The enzyme is pure according to polyacrylamide gel electrophoresis, has a molecular weight of 61,000-63,000 and consists of a single polypeptide chain. The enzyme is stable in air but not active. Activity is obtained only after complete removal of oxygen. EPR spectroscopy at 9 GHz shows a signal indicative for a [4Fe-4S]3+(3+,2+) cluster and in addition a rather complex signal of unknown origin. This additional signal completely disappears upon removal of oxygen, by incubation with 2-mercaptoethanol or by reduction with ferrocytochrome c. No EPR signals are detected in the enzyme reduced with H2 or dithionite. The intensity of the EPR signal of the [4Fe-4S] cluster corresponds to one-quarter of the enzyme concentration, both in the untreated as well as in the He- or N2-activated enzyme. If the enzyme is activated under He and then brought in contact with air the signal increases 4-fold and represents about one free spin/enzyme molecule. When measured at 35 GHz the line shape and peak positions of the additional signal change, indicating that the signal is not originating from a simple S = 1/2 system. None of the inhibitors of the hydrogenase activity has any effect on the shape or intensity o the EPR signal fo the Fe-S cluster, 2H2O also has no effect. All EPR signals disappear after reduction with NADH or ascorbate in the presence of phenazine methosulphate. It is suggested that the Fe-S cluster is not the primary site of interaction of H2 with the enzyme.
D002844:Chromatium / Q000201:enzymology*; D004578:Electron Spin Resonance Spectroscopy; D007506:Iron-Sulfur Proteins / Q000032:analysis; D008970:Molecular Weight; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D014461:Ultracentrifugation
D007506:Iron-Sulfur Proteins; D010088:Oxidoreductases