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

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-H)-NADPD by stopped-flow spectrophotometry. Mixing CtFNR 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 was mixed with excess NADP, the absorbance in the flavin band I increased to about 70 % of that of CtFNR with an apparent rate of ~4 s, 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.