Reaction of the NAD(P)H:flavin oxidoreductase from Escherichia coli with NADPH and riboflavin:: Identification of intermediates
Reaction of the NAD(P)H:flavin oxidoreductase from Escherichia coli with NADPH and riboflavin:: Identification of intermediates
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DOI：
10.1021/bi980396f
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发表时间：
1998-08-25
期刊：
影响因子：
2.9
通讯作者：
Fontecave, M
中科院分区：
文献类型：
--
作者：
Niviére, V;Vanoni, MA;Fontecave, M
黄素还原酶催化NAD(P)H还原游离黄素。作为分离的，大肠杆菌黄素还原酶不含任何黄素假基，但在氧化还原之前在三元配合物中容纳还原的吡啶核苷酸和黄素底物。用静态和快速动力学吸收光谱研究了黄素还原酶催化NADPH还原核黄素的过程。静态吸收光谱实验表明，在核黄素和还原吡啶核苷酸存在的情况下，黄素还原酶稳定NADP（+）和还原核黄素的电荷转移复合物，尽管在很小程度上稳定。此外，核黄素的还原基本上是不可逆的。快速动力学吸收光谱研究表明，在催化循环过程中存在两种具有长波长吸收的中间体。这些中间体具有类似于氧化黄素和NAD(P)H、还原黄素和NAD(P) +电荷转移配合物的光谱特性，这两种中间体已被鉴定为铁氧化还蛋白- nadp（+）还原酶家族中黄酶反应的中间体。由此，提出了一种黄素还原酶与NADPH和核黄素反应的最小动力学方案。在黄素还原酶与NADPH和核黄素形成Michaelis复合体后，形成第一个中间体，确定为NADPH和核黄素的电荷转移复合体。随后是酶结合NADP（+）和还原核黄素的第二电荷转移中间体。后者衰变，产生黄素还原酶与NADP（+）和还原核黄素的Michaelis复合体，然后核黄素解离完成反应。这些结果支持了黄素还原酶与铁氧化还原蛋白- nadp（+）还原酶家族在结构上相似的最初假设，并在功能水平上扩展了这种相似性。
Flavin reductase catalyzes the reduction of free flavins by NAD(P)H. As isolated, Escherichia coli flavin reductase does not contain any flavin prosthetic group but accommodates both the reduced pyridine nucleotide and the flavin substrate in a ternary complex prior to oxidoreduction. The reduction of riboflavin by NADPH catalyzed by flavin reductase has been studied by static and rapid kinetics absorption spectroscopies. Static absorption spectroscopy experiments revealed that, in the presence of riboflavin and reduced pyridine nucleotide, flavin reductase stabilizes, although to a small extent, a charge-transfer complex of NADP(+) and reduced riboflavin. In addition, reduction of riboflavin was found to be essentially irreversible. Rapid kinetics absorption spectroscopy studies demonstrated the occurrence of two intermediates with long-wavelength absorption during the catalytic cycle. Such intermediate species exhibit spectroscopic properties similar to those of charge-transfer complexes of oxidized flavin and NAD(P)H, and reduced flavin and NAD(P)(+), respectively, which have been identified as intermediates during the reaction of flavoenzymes of the ferredoxin-NADP(+) reductase family. Thus, a minimal kinetic scheme for the reaction of flavin reductase with NADPH and riboflavin can be proposed. After formation of the Michaelis complex of flavin reductase with NADPH and riboflavin, a first intermediate, identified as a charge-transfer complex of NADPH and riboflavin, is formed. It is followed by a second charge-transfer intermediate of enzyme-bound NADP(+) and reduced riboflavin. The latter decays, yielding the Michaelis complex of flavin reductase with NADP(+) and reduced riboflavin, which then dissociates to complete the reaction. These results support the initial hypothesis of a structural similarity between flavin reductase and the enzymes of the ferredoxin-NADP(+) reductase family and extend it at a functional level.