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

Seo, Daisuke  ,  Kitashima, Masaharu  ,  Sakurai, Takeshi  ,  Inoue, Kazuhito

xx ( xx )  , pp.xxxx - xxxx , 2016 , Springer Science+Business Media
Ferredoxin-NAD(P)+ oxidoreductase (FNR, [EC], [EC]) 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 CtFNRox 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 CtFNRred was mixed with excess NADP+, the absorbance in the flavin band I increased to about 70 % of that of CtFNRox 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. © 2016 Springer Science+Business Media Dordrecht

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