Studies on the one‐ and two‐electron FAD‐mediated reactions by ferrodoxin‐NADP+ oxidoreductase from Anabaena

The NADPH‐diaphorase activity of the flavoprotein ferredoxin‐NADP+ oxidoreductase from the cyanobacterium Anabaena sp. strain 7119 shows a very alkaline optimum pH value, about 9.5–10.0, when assayed with two‐electron acceptor dyes, such as dichlorophenolindophenol or iodonitrotetrazolium. In contrast, the enzyme reactions mediated by the physiological one‐electron carrier ferredoxin exhibit an almost neutral optimum pH value. EPR spectra and pH titration of the NADPH‐reduced enzyme indicate that whereas the one‐electron reduced form (semiquinone) of the flavin coenzyme is formed at neutral pH values, the fully reduced form (hydroquinone) arises at the alkaline range above pH 9.0. These findings are also in agreement with data obtained from redox titrations of the enzyme FAD by using an NADPH‐regenerating system and are interpreted on the basis of the hydroquinone and semiquinone forms of the flavin coenzyme being, respectively, intermediates in the diaphorase and ferredoxin‐mediated catalytic activities of the reductase.