Flavin-mediated reduction of ferric leghemoglobin from soybean nodules

The reduction of ferric leghemoglobin (Lb(3+)) from soybean (Glycine max (L.) Merr.) nodules by riboflavin, FMN and FAD in the presence of NAD(P)H was studied in vitro. The system NAD(P)H + flavin reduced Lb(3+) to oxyferrous (Lb(2+) · O2) or deoxyferrous (Lb(2+)) leghemoglobin in aerobic or anaerobic conditions, respectively. In the absence of O2 the reaction was faster and more effective (i.e. less NAD(P)H oxidized per mole Lb(3+) reduced) than in the presence of O2; this phenomenon was probably because O2 competes with Lb(3+) for reductant, thus generating activated O2 species. The flavin-mediated reduction of Lb(3+) did not entail production of superoxide or peroxide, indicating that NAD(P)H-reduced flavins were able to reduce Lb(3+) directly. The NAD(P)H + flavin system also reduced the complexes Lb(3+) · nicotinate and Lb(3+) · acetate to Lb(2+) · O2, Lb(2+) or Lb(2+) · nicotinate, depending on the concentrations of ligands and of O2. In the presence of 200 μM nitrite most Lb remained as Lb(3+) in aerobic conditions but the nitrosyl complex (Lb(2+) · NO) was generated in anaerobic conditions. The above-mentioned characteristics of the NAD(P)H + flavin system, coupled with its effectiveness in reducing Lb(3+) at physiological levels of NAD(P)H and flavins in soybean nodules, indicate that this mechanism may be especially important for reducing Lb(3+) in vivo.