K-062. The Recombinant Expression of a Soluble Oxidoreductase from Carboxydothermus ferrireducens

R. U. Onyenwoke;
Kenyon Coll., Gambier, OH.

Carboxydothermus ferrireducens is a thermophilic, dissimilatory iron-reducing, Gram-type positive bacterium that possesses multiple enzymes capable of reducing iron-containing compounds, e.g., Fe(III) (hydr)oxides and Fe(III) citrate. The mechanisms of Fe(III) reduction have been mainly studied in the mesophilic, Gram-type negative Proteobacteria. This fact leaves a void in mechanistic knowledge of Fe(III) reduction for distant lineages of microorganisms other than the Proteobacteria, e.g., Gram-type positive bacteria. Previously we reported on the purification and characterization of a soluble oxidoreductase from the cytoplasmic fraction of C. ferrireducens which is capable of the reduction of Fe(III); in addition to several other metals, including Mn(IV), Cr(VI), and Co(III); and quinones, including AQDS and menadione. The enzyme functions by a two-electron transfer mechanism. This type of mechanism is typically involved in cellular protection from reactive oxidative species formed from the products of one-electron transfers, i.e., semiquinones and in particular reactive species that can be generated from iron via Fenton chemistry. Recently, we have over-expressed and purified an N-terminal His-tagged version of this soluble oxidoreductase in an Escherichia coli host strain. Here we present our results on the over-expression and characterization of this recombinant protein.