Isolation and characterization of a soluble NADPH-dependent Fe(III) reductase from Geobacter sulfurreducens

NADPH is an intermediate in the oxidation of organic compounds coupled to F e(III) reduction in Geobacter species, but Fe(III) reduction with NADPH as the electron donor has not been studied in these organisms. Crude extracts of Geobacter sulfurreducens catalyzed the NADPH-dependent reduction of Fe(I II)-nitrilotriacetic acid (NTA), The responsible enzyme, which was recovere d in the soluble protein fraction, was purified to apparent homogeneity in a four-step procedure. Its specific activity for Fe(III) reduction was 65 m u mol.min(-1).mg(-1), The soluble Fe(III) reductase was specific for NADPH and did not utilize NADH as an electron donor, Although the enzyme reduced several forms of Fe(III), Fe(m)-NTA was the preferred electron acceptor. Th e protein possessed methyl viologen:NADP(+) oxidoreductase activity and cat alyzed the reduction of NADP(+) with reduced methyl viologen as electron do nor at a rate of 385 U/mg, The enzyme consisted of two subunits with molecu lar masses of 87 and 78 kDa and had a native molecular mass of 320 kDa, as determined by gel filtration. The purified enzyme contained 28.9 mol of Fe, 17.4 mol of acid-labile sulfur, and 0.7 mol of flavin adenine dinucleotide per mol of protein. The genes encoding the two subunits were identified in the complete sequence of the G. sulfurreducens genome from the N-terminal amino acid sequences derived from the subunits of the purified protein. The sequences of the two subunits had about 30% amino acid identity to the res pective subunits of the formate dehydrogenase from Moorella thermoacetica, but the soluble Fe(III) reductase did not possess formate dehydrogenase act ivity. This soluble Fe(III) reductase differs significantly from previously characterized dissimilatory and assimilatory Fe(III) reductases in its mol ecular composition and cofactor content.