Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16.
The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.
D000421:Alcaligenes / Q000201:enzymology*; D006859:Hydrogen; D007700:Kinetics; D008970:Molecular Weight; D009243:NAD; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet
D009243:NAD; D006859:Hydrogen; D010088:Oxidoreductases