Purification and properties of the membrane-bound hydrogenase from Proteus mirabilis.
The cytoplasmic membrane-bound hydrogenase of the facultative anaerobe, Proteus mirabilis, has been solubilized and purified to homogeneity. The purified enzyme exhibited a maximal specific activity of about 780 mumol H2 oxidized/min per mg protein (benzyl viologen reduction). The hydrogenase has a molecular weight of 205 000 and is composed of two subunits with a molecular weight of 63 000 and two of 33 000. The absorption spectrum of the enzyme was characteristic of non-heme iron proteins. The millimolar extinction coefficients at 400 and 280 nm are 106 and 390, respectively. The hydrogenase has about 24 iron atoms and 24 acid-labile sulfide atoms/molecule. Amino acid analyses revealed the presence of 39 half-cystine residues/molecule and a preponderance of acidic amino acids. The hydrogenase in its oxidized form exhibits an EPR signal of the HiPIP-type with g values at 2.025 and 2.018. Upon reduction with either dithionite or H2 the signal disappears; no other signals were detectable.
D000596:Amino Acids / Q000032:analysis; D002462:Cell Membrane / Q000201:enzymology*; D004227:Dithionite / Q000494:pharmacology; D004578:Electron Spin Resonance Spectroscopy; D006859:Hydrogen / Q000494:pharmacology; D007501:Iron / Q000032:analysis; D046911:Macromolecular Substances; D008970:Molecular Weight; D010088:Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D011511:Proteus / Q000201:enzymology*; D013440:Sulfides / Q000032:analysis
D000596:Amino Acids; D046911:Macromolecular Substances; D013440:Sulfides; D004227:Dithionite; D006859:Hydrogen; D007501:Iron; D010088:Oxidoreductases