Purification and properties of the membrane-bound hydrogenase from N2-fixing Alcaligenes latus.
The nitrogen-fixing, aerobic hydrogen-oxidizing bacterium Alcaligenes latus forms hydrogenase when growing lithoautotrophically with hydrogen as electron donor and carbon dioxide as sole carbon source or when growing heterotrophically with N2 as sole nitrogen source. The hydrogenase is membrane-bound and relatively oxygen-sensitive. The enzymes formed under both conditions are identical on the basis of the following criteria: molecular mass, mobility in polyacrylamide gel electrophoresis, Km value for hydrogen (methylene blue reduction), stability properties, localization, and cross-reactivity to antibodies raised against the 'autotrophic' hydrogenase. The hydrogenase was solubilized by Triton X-100 and deoxycholate treatment and purified by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose C1-4B, DEAE-Sephacel and Matrix Gel Red A under hydrogen to homogeneity to a specific activity of 113 mumol H2 oxidized/min per mg protein (methylene blue reduction). SDS gel electrophoresis revealed two nonidentical subunits of molecular weights of 67 000 and 34 000, corresponding to a total molecular weight of 101 000. The pure enzyme was able to reduce FAD, FMN, riboflavin, flavodoxin isolated from Megasphaera elsdenii, menadione and horse heart cytochrome c as well as various artificial electron acceptors. The reversibility of the hydrogenase function was demonstrated by H2 evolution from reduced methyl viologen.
D000421:Alcaligenes / Q000201:enzymology*; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D003574:Cytochrome c Group / Q000378:metabolism; D004579:Electron Transport; D006736:Horses; D006864:Hydrogenase; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D009206:Myocardium; D009586:Nitrogen Fixation; D010088:Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism
D003574:Cytochrome c Group; D046911:Macromolecular Substances; D010088:Oxidoreductases; D006864:Hydrogenase