The membrane‐bound hydrogenase from Paracoccus denitrificans

The membrane‐bound hydrogenase from Paracoccus dentitrificans was purified 68‐fold with a yield of 14.6%. The final preparation had a specific activity of 161.9 μmol H 2 min −1 (mg protein) −1 (methylene blue reduction). Purification involved solubilzation by Triton X‐114, phase separation, chromatography on DEAE‐Sephacel, ammonium‐sulfate precipitation and chromatography on Procion‐red HE‐3B‐Sepharose. Gel electrophoresis under denatruing conditions revealed two non‐identical subunits with molecular masses of 64 kDa and 34 kDa. The molecular mass of the native enzyme was 100kDa, as estimated by FPLC gel filtration in the presence of Chaps, a zwitterionic detergent. The isoelectric point of the Paracoccus hydrogenase was 4.3. Metal analysis of the purified enzyme indicated a content of 0.6 nickel and 7.3 iron atoms/molecule. ESR spectra of the reduced enzyme exhibited a close similarity to the membrane‐bound hydrogenase from Alcaligenes eutrophus H16 with g values of 1.86, 1.92 and 1.98. The half‐life for inactivation under air at 20 0 C was 8 h. The Praracoccus hydrogenase reduced several electron acceptors, namely methylene blue, benzyl viologen, methyl viologen, menadione, cytochrome c , FMN, 2,6‐dichloriondophenol, ferricyanide and phenazine methosulfate. The highest activity was measured with methylene blue ( V = 161.9 U/mg; K m = 0.04 mM), whereas benzyl and methyl viologen were reduced at distinctly lower rates (16.5 U/mg and 12.1 U/mg, respectively). The native hydrogenase from P. denitrificans cross‐reacted with purified antibodies raised aginst the membrane‐bound hydrogense from A. eutrophus H16. The corresponding subunits from both enzymes also showed immunological relationship. All reactions were of partial identity.