Kinetics of NADP.sup.+/NADPH reduction-oxidation catalyzed by the ferredoxin-NAD.sup.+ reductase from the green sulfur bacterium Chlorobaculum tepidum

• Published in: Photosynthesis research Vol. 130; no. 1-3; p. 479
• Main Authors: Seo, Daisuke, Kitashima, Masaharu, Sakurai, Takeshi, Inoue, Kazuhito
• Format: Journal Article
• Language: English
• Published: Springer 01.12.2016
• Subjects: Bacteria; Sulfur; Sulfur compounds; Thioredoxins
• ISSN: 0166-8595; 1573-5079
• DOI: 10.1007/s11120-016-0285-3

Ferredoxin-NAD(P).sup.+ 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.sup.+, NADPH, and (4S-.sup.2H)-NADPD by stopped-flow spectrophotometry. Mixing CtFNR.sub.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.sub.red was mixed with excess NADP.sup.+, the absorbance in the flavin band I increased to about 70 % of that of CtFNR.sub.ox with an apparent rate of ~4 s.sup.-1, whereas almost no absorption changes were observed in the CT band. Obtained data suggest that the reaction between CtFNR and NADP.sup.+/NADPH is reversible, in accordance with its physiological function.