Open AccessSelective release and function of one of the two FMN groups in the cytoplasmic NAD + ‐reducing [NiFe]‐hydrogenase from Ralstonia eutropha
Author(s) -
van der Linden Eddy,
Faber Bart W.,
Bleijlevens Boris,
Burgdorf Tanja,
Bernhard Michael,
Friedrich Bärbel,
Albracht Simon P. J.
Publication year - 2004
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.2004.03984.x
Subject(s) - nad+ kinase , chemistry , hydrogenase , enzyme , ralstonia , flavin mononucleotide , dimer , stereochemistry , photochemistry , biochemistry , cofactor , organic chemistry
The soluble, cytoplasmic NAD + ‐reducing [NiFe]‐hydrogenase from Ralstonia eutropha is a heterotetrameric enzyme (HoxFUYH) and contains two FMN groups. The purified oxidized enzyme is inactive in the H 2 ‐NAD + reaction, but can be activated by catalytic amounts of NADH. It was discovered that one of the FMN groups (FMN‐a) is selectively released upon prolonged reduction of the enzyme with NADH. During this process, the enzyme maintained its tetrameric form, with one FMN group (FMN‐b) firmly bound, but it lost its physiological activity – the reduction of NAD + by H 2 . This activity could be reconstituted by the addition of excess FMN to the reduced enzyme. The rate of reduction of benzyl viologen by H 2 was not dependent on the presence of FMN‐a. Enzyme devoid of FMN‐a could not be activated by NADH. As NADH‐dehydrogenase activity was not dependent on the presence of FMN‐a, and because FMN‐b did not dissociate from the reduced enzyme, we conclude that FMN‐b is functional in the NADH‐dehydrogenase activity catalyzed by the HoxFU dimer. The possible function of FMN‐a as a hydride acceptor in the hydrogenase reaction catalyzed by the HoxHY dimer is discussed.