Directionality of the Reversible Reduction/Oxidation Reactions Catalyzed by Ferredoxin-NAD(P)H Oxidoreductases from Phototrophic and Heterotrophic Bacteria

Ferredoxin-NAD(P)+ oxidoreductases (FNRs) of photoautotroph Chlorobaculum tepidum (CtFNR) and heterotroph Bacillus subtilis (BsFNR) are homo-dimeric flavoprotein exhibiting a significant conservation of structural topology with bacterial NADPH-thioredoxin reductase. Although the redox reaction catalyzed by FNR is theoretically reversible, under the physiological conditions the direction of the reaction is assumed to be optimized toward either NADP+ reduction in photosynthetic process or NADPH oxidation in non-photosynthetic one. In this presentation the directionality of the redox reactions with NADP+/H and the structure-function relationships will be discussed based on the pre-steady-state kinetics. NADPH oxidation and NADP+ reduction reactions by BsFNR occurred with rapid formations of charge transfer complexes (CTCs) followed by the rate-limiting hydride transfer. The rate of the NADPH oxidation direction in the hydride transfer step was much faster than that of NADP+ reduction direction (500 s−1 vs. < 10 s−1), indicating NADPH oxidation was favored in BsFNR-catalyzing reaction. The deletions of the C-terminal amino acid residues almost did not affect the directionality. NADPH oxidation by CtFNR yielded a rapid formation of CTCs followedby the rate-limiting hydride transfer. The amount of reduced CtFNR at the equilibrium was in part even in the presence of ∼50-fold excess NADPH. NADP+ reduction by CtFNR provided no charge transfer band and the observed rate for the NADP+ reduction by CtFNR was rather slow (∼ 5 s−1), suggesting the formation of CTC was the rate-limiting. The hydride transfer rates in both directions were estimated to be comparable, indicating redox reaction with NADP+/H was reversible in the case of CtFNR. The depletion of the C-terminal residues turns out CtFNR to be NADPH oxidizing enzyme, suggesting that the C-terminal residues are required for tuning the redox properties of the FAD prosthetic group.