Localization and Solubilization of the Iron(III) Reductase of Geobacter sulfurreducens

Abstract

The iron(III) reductase activity of Geobacter sulfurreducens was determined with the electron donor NADH and the artificial electron donor horse heart cytochrome c. The highest reduction rates were obtained with Fe(III) complexed by nitrilotriacetic acid as an electron acceptor. Fractionation experiments indicated that no iron(III) reductase activity was present in the cytoplasm, that approximately one-third was found in the periplasmic fraction, and that two-thirds were associated with 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 solubilized from the membrane fraction with 0.5 M KCl showing that the iron(III) reductase was weakly bound to the membranes. In addition, solubilization of the iron(III) reductase from whole cells with 0.5 M KCl, without disruption of cells, indicated that the iron(III) reductase is a peripheral protein on the outside of the outer membrane. Redox difference spectra of KCl extracts showed the presence of c-type cytochromes which could be oxidized by ferrihydrite. Only one activity band was observed in native polyacrylamide gels stained for the iron(III) reductase activity. Excision of the active band from a preparative gel followed by extraction of the proteins and sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed the presence of high-molecular-mass, cytochrome-containing proteins in this iron(III) reductase activity band. From these experimental data it can be hypothesized that the iron(III) reductase of G. sulfurreducens is a peripheral outer membrane protein that might contain ac-type cytochrome.

Identification of proteins capable of metal reduction from the proteome of the Gram‐positive bacterium Desulfotomaculum reducens MI‐1 using an NADH‐based activity assay

“…Electrophoresis was performed at ambient temperature under constant voltage of 50 V for 10 min, to allow the proteins to enter the gel and salts to dissipate, followed by separation at 125 V for ∼2 h or until the dye front reached the bottom of the gel cassette. The developed gels were removed from the cassette, rinsed briefly in deionized water, and then an in-gel iron reduction activity assay was subsequently performed, designed by modifying an existing protocol (Gaspard et al, 1998). Gels were submerged in the iron reduction activity assay buffer (described above) with 0.5 mM Fe(III)-NTA for 40 min.…”

Exocellular electron transfer in anaerobic microbial communities

“…The biochemistry of iron reduction of Geobacter sulfurreducens was studied in detail. Cytochrome-dependent and NAD-dependent Fe(III) reductases have been purified and characterized, and evidence was provided that a porin-like protein and a special cytochrome c are important in the reduction of iron (Gaspard et al, 1998;Seeliger et al, 1998;Kaufmann and Lovley, 2001;Magnuson et al, 2001;Afkar et al, 2005;Kim et al, 2005). Yet, the exact mechanism of electron transfer from this bacterium to the insoluble electron acceptor is not clear.…”

Iron metabolism in anoxic environments at near neutral pH

“…Also in G. metallireducens ferric reductase activity is localized in the membrane fraction and cytochromes are thought to be involved in electron transfer to ferric iron oxides [27]. In G. sulfurreducens, ferric reductase is a peripheral protein in the outer face of the outer membrane, and its activity is associated with c-type cytochromes which are oxidized by ferrihydrite [28].…”