TAILIEUCHUNG - Báo cáo khóa học: Selective release and function of one of the two FMN groups in the cytoplasmic NAD + -reducing [NiFe]-hydrogenase from Ralstonia eutropha

The soluble, cytoplasmic NAD + -reducing [NiFe]-hydro-genase fromRalstonia eutrophais aheterotetrameric enzyme (HoxFUYH) and contains two FMN groups. The purified oxidized enzyme is inactive in the H2-NAD + reaction, but can be activated by catalytic amounts of NADH. It was discovered that one of the FMN groups (FMN-a) is selec-tively 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 H2. . | Eur. J. Biochem. 271 801-808 2004 FEBS 2004 doi Selective release and function of one of the two FMN groups in the cytoplasmic NAD -reducing NiFe -hydrogenase from Ralstonia eutropha Eddv van der Linden1 Bart W. Faber1 Boris Bleijlevens1 Tanja Buradorf2 Michael Bernhard2 . Barbel Friedrich2 and Simon P. J. Albracht1 1 Swammerdam Institute for Life Sciences Biochemistry University of Amsterdam the Netherlands 2Institut fur BiologieịMikrobiologie Humboldt-Universităt zu Berlin Berlin Germany The soluble cytoplasmic NAD -reducing NiFe -hydro-genase from Ralstonia eutropha is a heterotetrameric enzyme HoxFUYH and contains two FMN groups. The purified oxidized enzyme is inactive in the H2-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 H2. This activity could be reconstituted by the addition of excess FMN to the reduced enzyme. The rate of reduction of benzyl viologen by H2 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-dehydro-genase 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. Keywords flavin NAD -reducing NiFe -hydrogenase Ralstonia eutropha. The facultative lithoautotrophic Knallgas bacterium Rals-tonia eutropha H16 contains three different NiFe -hydro-genases a membrane-bound enzyme 1-3 a soluble cytoplasmic hydrogenase SH which reduces NAD 1 4 5 and a protein functional in a .

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