Involvement of glutamic acid 301 in the catalytic mechanism of ferredoxin-NADP+ reductase from Anabaena PCC 7119
Involvement of glutamic acid 301 in the catalytic mechanism of ferredoxin-NADP+ reductase from Anabaena PCC 7119
复制标题
DOI：
10.1021/bi971795y
复制
发表时间：
1998-03-03
期刊：
影响因子：
2.9
通讯作者：
Gómez-Moreno, C
中科院分区：
文献类型：
--
作者：
Medina, M;Martínez-Júlvez, M;Gómez-Moreno, C
Anabaena PCC 7119铁氧还蛋白- nadp（+）还原酶（FNR）的晶体结构表明，Glu301的羧酸基可能直接参与了FAD的异alloxazine部分与FNR底物（NADPH、铁氧还蛋白和黄氧还蛋白）之间的电子和质子转移的催化过程。为了评估这种可能性，通过将残基突变为丙氨酸来去除Glu301的羧酸盐。各种光谱技术（紫外-可见吸收、荧光和CD）表明突变蛋白折叠正常，没有发生显著的蛋白质结构重排。此外，突变体FNR与其底物的复杂形成几乎没有改变。然而，在Glu301Ala FNR的光还原过程中没有发现半醌的形成。此外，在电子转移到铁氧还蛋白的过程中，FNR半醌形成所必需的稳态活性明显受到突变的影响。快速瞬态动力学研究证实，去除301位羧酸盐会使铁氧还蛋白的电子转移过程的速率常数降低约40倍，而不会明显影响配合物的形成，从而干扰了FAD和铁硫簇之间电子转移过渡态的稳定。此外，该突变还改变了FNR与5'-去氮杂黄素半醌的非特异性反应、与黄伏氧素的电子转移反应以及酶的再氧化性能。这些结果清楚地表明，Glu301是FNR中电子转移的关键残基。
The crystal structure of Anabaena PCC 7119 ferredoxin-NADP(+) reductase (FNR) suggests that the carboxylate group of Glu301 may be directly involved in the catalytic process of electron and proton transfer between the isoalloxazine moiety of FAD and FNR substrates (NADPH, ferredoxin, and flavodoxin). To assess this possibility, the carboxylate of Glu301 was removed by mutating the residue to an alanine. Various spectroscopic techniques (UV-vis absorption, fluorescence, and CD) indicate that the mutant protein folded properly and that significant protein structural rearrangements did not occur. Additionally, complex formation of the mutant FNR with its substrates was almost unaltered. Nevertheless, no semiquinone formation was seen during photoreduction of Glu301Ala FNR. Furthermore, steady-state activities in which FNR semiquinone formation was required during the electron-transfer processes to ferredoxin were appreciably affected by the mutation. Fast transient kinetic studies corroborated that removal of the carboxylate at position 301 decreases the rate constant approximately 40-fold for the electron transfer process with ferredoxin without appreciably affecting complex formation, and thus interferes with the stabilization of the transition state during electron-transfer between the FAD and the iron-sulfur cluster. Moreover, the mutation also altered the nonspecific reaction of FNR with 5'-deazariboflavin semiquinone, the electron-transfer reactions with flavodoxin, and the reoxidation properties of the enzyme. These results clearly establish Glu301 as a critical residue for electron transfer in FNR.