Ferric leghemoglobin reductase from soybean root nodules

Arch Biochem Biophys. 1984 May 15;231(1):102-13. doi: 10.1016/0003-9861(84)90367-9.

Abstract

An NADH: (acceptor) oxidoreductase from the cytosol of soybean root nodules was purified by ammonium sulfate fractionation, hydroxylapatite adsorption, and Sephacryl S-200 Superfine chromatography. The native molecular weight of the reductase was found to be 100,000 by analytical gel filtration and 83,000 by equilibrium ultracentrifugation. The subunit molecular weight was 54,000 as determined by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The pI of the enzyme was 5.5. With ferric leghemoglobin (Lb) as the substrate, nearly identical initial velocities were obtained using either CO or O2 to ligate the enzymatically produced ferrous leghemoglobin. With CO as the ligand in the reaction, the product of the enzyme-catalyzed, NADH-dependent reduction of ferric Lb was spectrally identified as LbCO. Initial velocity was a linear function of increasing enzyme concentration. NADPH was only 31% as effective an electron donor as NADH as determined by initial velocity. The Michaelis constants (Km) for ferric Lba and NADH were 9.5 and 18.8 microM, respectively. Myoglobin, Lba, Lbc1, Lbc2, Lbc3, and Lbd were reduced at similar rates by the reductase. At pH 5.2, acetate-bound ferric Lb and nicotinate-bound ferric Lb were reduced by the enzyme at 83 and 5%, respectively, of rates observed in the absence of these ligands. The rate of enzymatic reduction of ferric Lb was constant between pH 6.5 and 7.6 but increased approximately threefold at pH 5.2. The results indicate that the NADH: (acceptor) oxidoreductase could be identified as a ferric Lb reductase.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Catalysis
  • Chemical Phenomena
  • Chemistry
  • Cytosol / enzymology
  • Glycine max / enzymology*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Weight
  • NADH, NADPH Oxidoreductases / isolation & purification
  • Substrate Specificity

Substances

  • NADH, NADPH Oxidoreductases
  • ferric leghemoglobin reductase