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

Abstract

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-2H)-NADPD by stopped-flow spectrophotometry. Mixing CtFNRox with NADPH yielded a rapid decrease of the absorbance in flavin band I centered at 460nm within 1ms, 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 600nm increased rapidly within 1ms, 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 ~4s-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.