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

Authors

Manuel Becana, University of Nebraska-Lincoln
Robert V. Klucas, University of Nebraska - Lincoln

Document Type

Article

Date of this Version

1990

Comments

Published in Proc. Natl. Acad. Sci. USA Vol. 87, pp. 7295-7299, September 1990

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 and exhibits a K_m of 9 µM for soybean Lb³⁺ component a and a K. 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 N0₂ ˉ. (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 0₂ˉ or H₂0₂, 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 0₂ˉ.