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

• Published in: Photosynthesis research Vol. 130; no. 1-3; pp. 479 - 489
• Main Authors: Seo, Daisuke, Kitashima, Masaharu, Sakurai, Takeshi, Inoue, Kazuhito
• Format: Journal Article
• Language: English
• Published: Dordrecht Springer Netherlands 01.12.2016
• Subjects: Biochemistry; Biomedical and Life Sciences; Chlorobium - enzymology; Chlorobium - metabolism; Ferredoxin-NADP Reductase - metabolism; Kinetics; Life Sciences; NADP - metabolism; Original Article; Oxidation-Reduction; Plant Genetics and Genomics; Plant Physiology; Plant Sciences; Protein Structure, Tertiary; Spectrophotometry - methods; Kinetic isotope effect; Charge transfer complex; Stopped flow; Flavoenzyme; Adrenodoxin; Putidaredoxin
• ISSN: 0166-8595; 1573-5079
• DOI: 10.1007/s11120-016-0285-3

Ferredoxin-NAD(P) + oxidoreductase (FNR, [EC 1.18.1.2], [EC 1.18.1.3]) from the green sulfur bacterium Chlorobaculum tepidum ( Ct FNR) is a homodimeric flavoprotein with significant structural homology to bacterial NADPH-thioredoxin reductases. Ct FNR homologs have been found in many bacteria, but only in green sulfur bacteria among photoautotrophs. In this work, we examined the reactions of Ct FNR with NADP + , NADPH, and (4 S - 2 H)-NADPD by stopped-flow spectrophotometry. Mixing Ct FNR 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 Ct FNR red was mixed with excess NADP + , the absorbance in the flavin band I increased to about 70 % of that of Ct FNR 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 Ct FNR and NADP + /NADPH is reversible, in accordance with its physiological function.