Reduction of ferric leghemoglobin in soybean root nodules

Reduction of ferric leghemoglobin to ferrous leghemoglobin in soybean nodules (Glycine max [L.] Merr. cv Woodworth) was studied using a spectrophotometer equipped with an in-cell space diffuse reflectance accessory. Nodule slices prepared and scanned under nitrogen gas showed a ferrous leghemoglobin absorption spectrum. Nodule slices equilibrated with 100% O(2) or air exhibited two absorption bands characteristic of oxygenated leghemoglobin. The addition of CO shifted those bands to CO leghemoglobin absorption bands. Potassium ferricyanide was not effective in oxidizing ferrous to ferric leghemoglobin in nodule slices. However, ferric leghemoglobin was formed by treating the nodule slices with hydroxylamine, and this was confirmed by complexing the ferric leghemoglobin to acetate, fluoride, or nicotinic acid. The diminution of ferric leghemoglobin was monitored as a function of time, and in the presence of nicotinic acid, the conversion of ferric to ferrous leghemoglobin was monitored by the appearance of ferrous leghemoglobin nicotinate complex as a function of time. Ferric leghemoglobin reduction was also confirmed by direct transmission spectrophotometry. The evidence presented here suggests that ferrileghemoglobin reduction occurs in nodule slices.