LEGHEMOGLOBIN REDUCTASE FROM SOYBEAN NODULES

Leghemoglobin (Lb) is a monomeric, oxygen-binding hemoprotein found in the root nodules of legumes. The heme iron of Lb must be in the ferrous state for Lb to remain functional (ie., to facilitate oxygen diffusion). A protein (Lb reductase) capable of catalyzing the reduction of ferric Lb in vitro from soybean (Glycine max L. Merr.) root nodules was purified over 1000-fold by ammonium sulfate fractionation, hydroxylapatite adsorption and Sephacryl S-200 Superfine chromatography. The purification of Lb, the substrate for Lb reductase, required an additional Sephadex G-75 chromatography step to remove contaminating Lb reductase. The native molecular weight of Lb reductase was found to be 100,000 daltons by analytical gel filtration, 83,000 daltons by equilibrium ultracentrifugation and 54,000 daltons by SDS-polyacrylamide slab gel electrophoresis. The pI of the enzyme was 5.5. Nearly identical initial velocities were obtained using either CO or O(,2) to ligate the enzymatically produced Lb(\u27+2). With CO as the ligand in the reaction, the product of the enzyme-catalyzed, NADH-dependent reduction of Lb(\u27+3) was spectrally identified as LbCO. Initial velocity was a linear function of increasing protein concentration. NADPH was only 31 percent as effective an electron donor as NADH. The Michaelis constants (K(,m)) for Lba(\u27+3) and NADH were 9.5 (mu)M and 18.8 (mu)M, respectively. Myoglobin, Lba, LBc(,1), Lbc(,2), Lbc(,3), and Lbd were reduced at similar rates by Lb reductase. Dichlorophenolindophenol was also reduced by the enzyme but dichlorophenolinophenol reduction was not a good criterion for purification. Methylene blue (1.0 (mu)M) stimulated the enzymatic reduction 10-fold. At pH 5.2, acetate-bound Lb(\u27+3) and nicotinate-bound Lb(\u27+3) were reduced by Lb reductase at 83 percent and 5 percent, respectively, of rates observed in the absence of ligand. The rate of the enzymatic reduction of Lb(\u27+3) was constant between pH 6.5 and 7.6 but increased approximately 3-fold at pH 5.2 Nodule homogenates contained a heat-stable, low molecular weight molecule (\u3c 5000 daltons) capable of reducing Lb(\u27+3) in the presence of NADH