Studies on the reaction mechanism of NADPH-adrenodoxin reductase with NADPH

The existence of a multi-component equilibrium involving a reversible reaction was demonstrated in the anaerobic reaction of NADPH-adrenodoxin reductase or ferredoxin-NADPH reductase; EC 1.18.1.2 (AdR) with NADPH or in the photoreduction of AdR in the presence of NADP+ and EDTA. After anaerobic reduction of AdR with excess NADPH, not only the fully reduced state but also other states of the enzyme were demonstrated as a mixture of characteristic absorption spectra of a flavin semiquinone with a peak at 575 nm and a shoulder around 625 nm and a charge transfer complex between NADP+ and the fully reduced AdR with a broad absorbance in a longer wavelength region. The amount of oxidized form in the reaction mixture was only a trace judging from the absorbance at 450 nm. The oxidized form was observed in addition to the other spectral species when the concentration of NADPH was limited. The spectrum of the AdR reduced with excess NADPH changed reversibly between 2 degrees C and 29 degrees C with isosbestic points at 427 nm and 667 nm; the semiquinone form increased at the expense of the charge transfer complex at lower temperatures, and vice versa at higher temperatures. The anaerobic photoreduction of AdR in the presence of NADP+ and EDTA produced the same spectral species as seen in the NADPH-reduced AdR, and the spectrum of the photoreduced AdR also changed reversibly between 1 degree C and 33 degrees C in a similar way to that of the NADPH-reduced AdR; the oxidized form changed in parallel with the semiquinone form. It was also found that NADPH was produced continuously during the photoirradiation even after the spectral changes had ceased. The results show that anaerobic reduction of AdR with NADPH or the photoreduction of AdR in the presence of NADP+ and EDTA produces a multi-component equilibrium and the equilibrium is dependent upon the concentration of NADPH and the temperature. Attainment of the equilibrium involves not only the forward reaction of AdR with NADPH but also the reverse reaction of the fully reduced AdR with NADP+.