LOCALIZATION AND SOLUBILIZATION OF THE IRON(III) REDUCTASE OF GEOBACTER-SULFURREDUCENS

The iron(III) reductase activity of Geobacter sulfurreducens was deter mined with the electron donor NADH and the artificial electron donor h orse heart cytochrome c. The highest reduction rates were obtained wit h Fe(III) complexed by nitrilotriacetic acid as an electron acceptor, Fractionation experiments indicated that no iron(III) reductase activi ty was present in the cytoplasm, that approximately one-third was foun d in the periplasmic fraction, and that two-thirds were associated wit h the membrane fraction, Sucrose gradient separation of the outer and cytoplasmic membranes showed that about 80% of the iron(III) reductase was present in the outer membrane, The iron(III) reductase could be s olubilized from the membrane fraction with 0.5 M KCI showing that the iron(III) reductase was weakly bound to the membranes, In addition, so lubilization of the iron(III) reductase from whole cells with 0.5 M KC I, without disruption of cells, indicated that the iron(III) reductase is a peripheral protein on the outside of the outer membrane. Redox d ifference spectra of KCI extracts showed the presence of c-type cytoch romes which could be oxidized by ferrihydrite, Only one activity band was observed in native polyacrylamide gels stained for the iron(III) r eductase activity, Excision of the active band from a preparative gel followed by extraction of the proteins and sodium dodecyl sulfate-poly acrylamide gel electrophoresis revealed the presence of high-molecular -mass, cytochrome-containing proteins in this iron(III) reductase acti vity band. From these experimental data it can be hypothesized that th e iron(III) reductase of G, sulfurreducens is a peripheral outer membr ane protein that might contain a c-type cytochrome.