Characterization of NADH-dependent Fe3+-chelate reductases of maize roots

Abstract

Iron-deficient maize seedlings exhibit a starvation syndrome characterized by an increase in different parameters such as root fresh weight (+ 30%), protein (+ 25%) and plasma membrane-associated NADH Fe3+ −EDTA reductase (NFR; +45%). NFR activity was found associated with 9 000g (20 min) and 110 000 g (1 h) sediments, purified plasma membrane and 110000 g supernatants. No differences were observed between the properties of reductases from Fe-starved versus Fe-sufficient roots. The characterization of NFR was undertaken. Low Mr forms (46 and 28 kDa, as detected by size-exclusion chromatography) were present in all fractions whereas 210 and 110 kDa forms were unique in membranes and 110 000 g supernatants, respectively. The 210 kDa form was solubilized from microsomes and characterized. The enzyme is cetone-resistant and appears to be comprised largely if not totally of the low Mr forms (46 and 28 kDa, corresponding to 30 and 32 kDa bands, respectively, in SDS-PAGE). The 210 kDa form tended to break down to subunits following dilution, and the effect could be prevented by addition of 10% (v/v) glycerol. A three-step purification procedure for microsomal NFR was devised, consisting of acetone fractionation of lysophosphatidycholine solubilized microsomes, Blue Sepharose CL-6B affinity chromatography and a final size exclusion chromatography in the absence of detergent, resulting in a 700-fold purification of the 28 kDa protein. The best electron acceptor for the purified 28 kDa form was ferricyanide (400μmol min−1mg−1 protein) followed by Fe3+−chelates (up to 200μol min−1 mg−1 protein) and other compounds to a lesser extent (cyt c, DCPIP).The 46 kDa form, on the other hand, had high ferricyanide reductase activity (about 300μmol min−1mg−1 protein) and relatively low Fe3+−chelate reductase activity. The properties of NFR (high M, active forms, donor and acceptor specificity, purification behaviour, large hydrophilic domains, size of subunits) suggest a relationship with the NADH-cyt b5 reductase family of FAD-containing proteins. None of the latter flavoproteins is a transmembrane enzyme.