Kinetics of NADP⁺/NADPH reduction–oxidation catalyzed by the ferredoxin-NAD(P)⁺ reductase from the green sulfur bacterium Chlorobaculum tepidum

详细信息    查看全文

• 作者：Daisuke Seo ; Masaharu Kitashima ; Takeshi Sakurai ; Kazuhito Inoue
• 关键词：Flavoenzyme ; Adrenodoxin ; Putidaredoxin ; Stopped flow ; Charge transfer complex ; Kinetic isotope effect
• 刊名：Photosynthesis Research
• 出版年：2016
• 出版时间：December 2016
• 年：2016
• 卷：130
• 期：1-3
• 页码：479-489
• 全文大小：1,299 KB
• 刊物类别：Biomedical and Life Sciences
• 刊物主题：Life Sciences
  Plant Physiology
  
• 出版者：Springer Netherlands
• ISSN：1573-5079
• 卷排序：130

文摘

Ferredoxin-NAD(P)+ oxidoreductase (FNR, [EC 1.18.1.2], [EC 1.18.1.3]) from the green sulfur bacterium Chlorobaculum tepidum (CtFNR) is a homodimeric flavoprotein with significant structural homology to bacterial NADPH-thioredoxin reductases. CtFNR homologs have been found in many bacteria, but only in green sulfur bacteria among photoautotrophs. In this work, we examined the reactions of CtFNR with NADP+, NADPH, and (4S-2H)-NADPD by stopped-flow spectrophotometry. Mixing CtFNR_ox with NADPH yielded a rapid decrease of the absorbance in flavin band I centered at 460 nm within 1 ms, and then the absorbance further decreased gradually. The magnitude of the decrease increased with increasing NADPH concentration, but even with ~50-fold molar excess NADPH, the absorbance change was only ~45 % of that expected for fully reduced protein. The absorbance in the charge transfer (CT) band centered around 600 nm increased rapidly within 1 ms, then slowly decreased to about 70 % of the maximum. When CtFNR_red was mixed with excess NADP+, the absorbance in the flavin band I increased to about 70 % of that of CtFNR_ox with an apparent rate of ~4 s−1, whereas almost no absorption changes were observed in the CT band. Obtained data suggest that the reaction between CtFNR and NADP+/NADPH is reversible, in accordance with its physiological function.