van der Linden Eddy, Faber Bart W, Bleijlevens Boris, Burgdorf Tanja, Bernhard Michael, Friedrich Bärbel, Albracht Simon P J
Swammerdam Institute for Life Sciences, Biochemistry, University of Amsterdam, The Netherlands.
Eur J Biochem. 2004 Feb;271(4):801-8. doi: 10.1111/j.1432-1033.2004.03984.x.
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 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-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.
来自真养产碱菌的可溶性胞质NAD⁺还原型[NiFe]氢化酶是一种异源四聚体酶（HoxFUYH），含有两个FMN基团。纯化的氧化态酶在H₂-NAD⁺反应中无活性，但可被催化量的NADH激活。研究发现，在用NADH长时间还原该酶后，其中一个FMN基团（FMN-a）会被选择性释放。在此过程中，该酶保持其四聚体形式，有一个FMN基团（FMN-b）牢固结合，但失去了其生理活性——即H₂还原NAD⁺的活性。通过向还原态酶中添加过量FMN可恢复此活性。H₂还原苄基紫精的速率不依赖于FMN-a的存在。不含FMN-a的酶不能被NADH激活。由于NADH脱氢酶活性不依赖于FMN-a的存在，且因为FMN-b不会从还原态酶上解离，我们得出结论，FMN-b在由HoxFU二聚体催化的NADH脱氢酶活性中起作用。本文讨论了FMN-a在由HoxHY二聚体催化的氢化酶反应中作为氢化物受体的可能功能。
