Ferredoxins from Nitrogen-Fixing Bacteria

Two ferredoxins have been purified from N2-fixing Mycobacterium flavum. Ferredoxin I had a minimum molecular weight (by amino acid analysis) of 11 230 and contained 7.4 mol Fe and 7.0 mol S2per mol. The ratio of the absorbance at 400 nm to that at 275 nm was 0.7. Ferredoxin I1 had a minimum molecular weight of 13478 and contained 3.4 mol Fe and 3.2 mol S2per mol. The ratio of the absorbance at 400 nm to 268 nm was 0.42. Both ferredoxins were paramagnetic when oxidised by potassium ferricyanide and diamagnetic when reduced by sodium dithionite. This suggests that, like Azotobacter ferredoxin, they operate solely between the 2 and 1 oxidation states. A ferredoxin fraction of similar properties was isolated from Spirillum lipoferum. The electron paramagnetic resonance (EPR) spectra of both ferredoxins had features at g = 2.023, 2.017 and 2.00. The signals were affected by pH: below pH 6.0 that at g = 2.023 predominated, whereas above pH 6.0 the g = 2.017 feature was clearly present. The g = 2.00 feature was broader between pH 6.0 and 9.0 than outside this pH range. The change to the low-pH form was not reversible. Ferredoxin I had redox centres with mid-point potentials at 420 mV and + 230 mV; ferredoxin I1 had one redox centre of mid-point potential at approximately 420 mV. Above + 300 mV ferredoxin I suffered irreversible oxidative damage and lost the g = 2.023 feature of the EPR spectrum. Both ferredoxins were damaged by prolonged exposure to potassium ferricyanide. Ferredoxin I took up 1.8 electrons per molecule when reduced by sodium dithionite but the EPR signal of the oxidised form integrated to only 0.35 electron per molecule. Ferredoxin I1 took up 0.9 electron per molecule on reduction and the EPR signal of the oxidised form integrated to 0.66 electron per molecule. It was concluded that M.flavum ferredoxin I resembles Azotobacter ferredoxin and is thus the second ferredoxin isolated that has two redox centres with mid-point potentials approximately 700 mV apart. Ferredoxin I1 has no exact counterpart in other systems. Aerobic nitrogen-fixing bacteria apparently contain ferredoxins which operate between the 2 and 1 oxidation states.