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-2)-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 similar to 50-fold molar excess NADPH, the absorbance change was only similar to 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 similar to 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.