The purification and properties of nitrite reductase from higher plants, and its dependence on ferredoxin

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Abstract
NADPH-dependent nitrite reductase from the leaves of higher plants was purified at least 70-fold and separated into 2 enzyme fractions. The first enzyme, a diaphorase with ferredoxin-NADP-reductase activity, is required only to transfer electrons from NADPH to a suitable electron acceptor, which then donates electrons to nitrite reductase proper. Purified nitrite reductase accepted electrons from ferredoxin (the natural donor) or from reduced dyes. Ferredoxin was reduced by illuminated chloroplasts or dithlonite, or by NADPH when diaphorase was present. The purified enzyme did not accept electrons directly from NADPH. Ferredoxins purified from maize, spinach or Clostridium were interchangeable in the nitrite-reductase system. Nitrite reductase had Km 0.15 mil for nitrite. The pH optimum varied with plant and method of assay. The preparation had low sulphite-reductase activity. Ammonia was the product of nitrite reduction. For some plants, the assay of crude preparations with NADPH was limited by diaphorase and the addition of diaphorase gave a better estimate of nitrite-reductase activity. A simple method of assay is described that uses dithionite with benzyl viologen as electron donor.