Enzymatic and nonenzymatic mechanisms for ferric leghemoglobin reduction in legume root nodules

Abstract

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 andexhibits a Km of 9 microM for soybean LB3+ component a and a Km of 51 microM 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 Vmax values. The reductase is inactive with 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.-.