Functional characterization of the FoxE iron oxidoreductase from the photoferrotroph Rhodobacter ferrooxidans SW2

Abstract

Photoferrotrophy is presumed to be an ancient type of photosynthetic metabolism in which bacteria use the reducing power of ferrous iron to drive carbon fixation. In this work the putative iron oxidoreductase of the photoferrotroph Rhodobacter ferrooxidans SW2 was cloned, purified, and characterized for the first time. This protein, FoxE, was characterized using spectroscopic, thermodynamic, and kinetic techniques. It is a c-type cytochrome that forms a trimer or tetramer in solution; the two hemes of each monomer are hexacoordinated by histidine and methionine. The hemes have positive reduction potentials that allow downhill electron transfer from many geochemically relevant ferrous iron forms to the photosynthetic reaction center. The reduction potentials of the hemes are different and are cross-assigned to fast and slow kinetic phases of ferrous iron oxidation in vitro. Lower reactivity was observed at high pH and may contribute to prevent ferric iron precipitation inside or at the surface of the cell. These results help fill in the molecular details of a metabolic process that likely contributed to the deposition of precambrian banded iron formations, globally important sedimentary rocks that are found on every continent today.

FIGURE 1.

SDS-PAGE showing pure FoxE. The UV-visible spectrum of FoxE as purified is shown. The inset shows the fully reduced (black) and fully oxidized (gray) spectra of FoxE in the α and β region.

FIGURE 2.

X-band EPR spectrum of fully oxidized FoxE with the background from the cavity subtracted. At the top the g values of the main features of the spectrum are indicated. mT, millitesla.

FIGURE 3.

One-dimensional ¹H NMR spectra of FoxE poised at different degrees of oxidation from fully oxidized (bottom) to fully reduced (top). Signals identified with a circle belong to the HP heme, and signals identified with a square belong to the LP heme. The rounded box at approximately −3 ppm highlights the signals tentatively assigned to the heme axial methionines.

FIGURE 4.

One-dimensional ¹H NMR spectra of FoxE fully re-oxidized with K₃Fe(CN)₆ after reduction with sodium dithionite (top) or sodium ascorbate (bottom). Full oxidation of both samples was independently confirmed by UV-visible spectroscopy.

FIGURE 5.

Redox titrations of FoxE at pH 6 and 7 at 25 °C. The solid lines represent the best fits of the data. SHE, standard hydrogen electrode.

FIGURE 6.

Kinetic traces of FoxE reduction with Fe(II)-EDTA at pH 6 and 7 at 25 °C. At each pH FoxE was reduced starting from totally or partially oxidized. The solid lines represent the best fit of the data.

FIGURE 7.

Electron flow diagram in the periplasm of SW2. The vertical bar represents reduction potential. The solid arrows indicate electron flow. FoxE can potentially reduce any of the components of the cyclic photosynthesis electron chain: bc₁, cytochrome bc₁; c₂, cytochrome c₂; RC, photosynthetic reaction center. The dashed arrow indicates the electron transfer through the quinone pool from the light excited RC to the cytochrome bc₁. The vertical Fe²⁺ line indicates the reduction potential range of Fe²⁺ species at circumneutral pH.

FIGURE 8.

Schematic representation of reduction of each FoxE monomer. Open circles represent oxidized heme, and closed circles represent reduced hemes. Indicated are the reduction rate constants present in Table 2. A, shown are conditions where the cytochrome is fully oxidized at the beginning of the kinetic experiment. The dashed lines indicate slow intramonomer electron transfer. Because K_LP is faster than the intramonomer electron transfer, the bottom microstate has a negligible contribution in the kinetic experiment. B, shown are conditions when the cytochrome is partially reduced at the beginning of the kinetic experiment. The system has reached equilibrium between the two intermediate microstates before the reaction with Fe(II)-EDTA is initiated.