ENZYMATIC AND NONENZYMATIC MECHANISMS FOR FERRIC LEGHEMOGLOBIN REDUCTION IN LEGUME ROOT-NODULES

Evidence is presented for the operation in nodules of at least four systems for restoring functional ferrous leghemoglobin (Lb2+) from its inactive, ferric form. (i) Reduction of ferric leghemoglobin (Lb3+) by a reductase. The enzyme is a flavoprotein of 100 kDa with two equally sized subunits and exhibits aK(m) of 9 μM for soybean Lb3+ component a and aK(m) of 51 μM for NADH. NADPH is only 30% (initial velocities) as effective as NADH. Lb3+ reductase converts 215 nmol of Lb3+ to Lb2+·CO (or Lb2+·O2) per mg of protein per min and does not require an exogenous electron carrier. The enzyme shows similar affinity for soybean, bean, and cowpea Lb3+, but different V(max) values. The reductase is inactive when Lb3+ is bound to nicotinate or NO2-. (ii) Direct reduction of Lb3+ by NAD(P)H, ascorbate, and cysteine. Reduction by NAD(P)H is greatly stimulated by trace amounts of metals such as Mn2+. (iii) Reduction of Lb3+ by the flow of electrons from NAD(P)H to free flavins to Lb3+. The reaction does not occur via O2-/· or H2O2, and thus NAD(P)H-reduced flavins can directly reduce Lb3+. The efficiency of the reaction follows the order riboflavin > FMN > FAD. (iv) Reduction of Lb3+ by an unknown compound, B, of nodules. B has a molecular mass < 1 kDa and is heat-stable. The reaction mediated by B differs from those mediated by flavins and metals in several ways, requires NAD(P)H, and generates O2-/·.