Electron exchange between ferredoxin, cytochrome, and hematite
C.M. Eggleston1, N.Khare1, P.J.S.Colberg2
1
Department of Geology and Geophysics, 2Department of Zoology and Physiology, University of Wyoming,
Laramie, Wyoming, USA
G.Jordan3, M.Etienne4
3
Institute of Geology, Mineralogy and Geophysics, 4Institute of Chemistry, Ruhr Universitaet Bochum,
Bochum, Germany
ABSTRACT: The use of solid ferric hydr(oxides) as respiratory electron acceptors by dissimilatory iron reducing bacteria imposes constraints on the properties of molecules involved in the process. Several metalloproteins change redox properties when sorbed on electrode surfaces. Sorption-induced folding and orientation
changes may be involved in electron transport to mineral surfaces. Here, we present initial results for the interaction of ferredoxin (an iron sulfur protein) and c-type cytochrome (a heme-based protein) with hematite
surfaces. The results imply that the redox properties of ferredoxin depend on its sorbed concentration, the
presence of other adsorbates (impurities as well as cytochrome), and pH. This is consistent with observations
of the properties of these biomolecules on non-mineral electrodes.
1 INTRODUCTION
It has been known for well over a decade that a
number of species of Bacteria and Archaea can couple the reduction of FeIII and other metals to the oxidation of organic matter. Indeed, this respiratory
strategy may have been among the first to evolve
(e.g. Lovley 2002). These organisms have been the
subject of growing interest because of their novel
electron transport pathways and their bioremediation
potential. In order to understand and optimize the
usefulness of these organisms, it is necessary to understand the pathways by which they transfer electrons to FeIII and other metals. Electron transfer to a
solid mineral presents biochemical challenges that
differ from those encountered in the use of gasses
(or dissolved gasses) as respiratory electron acceptors. Some species of dissimilatory iron reducing
bacteria (DIRB) apparently require direct contact
with ferric minerals in order to accomplish iron reduction (e.g. Nevin & Lovley 2000), while others
appear to have the ability to synthesize soluble
“electron shuttle” molecules (although this is controversial; e.g. Newman & Kolter, 2000; Hernandez
& Newman, 2001).
Among the molecules implicated in the outermembrane electron transport chain used in FeIII reduction are quinones (e.g., menaquinone), c-type cytochromes , and possibly melanin (Myers & Myers
1993, 1997; Newman & Kolter 2000; Turick et al.
2002). Several DIRB species can reduce ferric minerals when provided with electron shuttle molecules
such as anthraquinone-2,6-disulfonate (AQDS) and
humic acids (e.g. Lovley et al. 1996; Zachara et al.
1998). Here, we report initial steps taken toward
understanding whether another molecule, iron-sulfur
protein (ferredoxin), could act as an electron shuttle
between cytochromes (associated with the outer
membrane of DIRB) and an iron oxide surface.
Wirtz et al. (2000) have shown that ferredoxin can
act as an electron shuttle between indium-doped tin
oxide (ITO) electrodes and cytochrome P450.
The [2Fe-2S] iron sulfur protein (ferredoxin) of
the well-studied and ubiquitous cytochrome bc1
complex (e.g. Crofts & Berry 1998) has recently
been found to mechanically transport electrons from
a reduction site to an oxidation site through redoxinduced changes in molecular orientation and shape,
resulting in large-scale domain movement within the
bc1 complex (Darrouzet et al. 2002; Darrouzet &
Daldal 2003). Such a redox-induced “extensionretraction” electron transport mechanism offers an
alternative in the controversy over whether the last
step in iron reduction by DIRB involves a soluble,
mobile electron shuttle molecule or instead requires
direct cell-mineral contact. Iron sulfur proteins have
been found to be part of the ability of some DIRB to
reduce iron (Kaufmann & Lovley 2001), along with
cytochromes and quinones.
Our goal is to investigate a similar system to that
of Wirtz et al. (2002), substituting hematite for the
ITO and focusing on the ability of ferredoxin to
shuttle electrons between cytochome (which would
be associated with DIRB outer membrane) and the
hematite surface (which would likely be separated
from the outer membrane by a lipopolysaccharide
layer). This is a preliminary report presenting some
AFM results for c-type horse heart cytochrome
sorbed to hematite, and cyclic voltammetry results
for ferredoxin, and ferredoxin together with cytochrome, on hematite. AFM of ferredoxin, voltammetry of cytochrome on hematite, and corresponding
adsorption studies the subject of ongoing work.
2 METHODS
Natural n-type hematite with a charge carrier density
of about 2.0 x 10-3 atom% due to Sn and Ti impurities was used (see Eggleston et al. 2003). Horse
heart cytochrome c was purchased from Sigma and
dissolved in 1 M ammonium phosphate at a concentration of 0.3 mg L-1. Atomic force microscope
(AFM) imaging was accomplished both by evaporating a droplet of cytochrome solution onto a hematite
sample, and by in-situ imaging in a diluted (0.03 mg
L-1) solution. Spinach ferredoxin was purchased
from Sigma in a liquid (35% solution) 0.15M
Trizma and NaCl buffer, pH 7.5. Hematite crystals
were immersed in in MOPS buffer at pH 7, and the
ferredoxin solution injected as received (MOPS
buffer was used in similar cyclic voltammetry experiments by Wirtz et al. 2002) to give a solution
concentration of 0.5 mg L-1. Subsequent additions
of ferredoxin solution gave concentrations of 0.88
and 1.1 mg L-1. The solution pH was checked after
each change of aqueous conditions, including acidifications with HCl. AFM was conducted using a
Digital Instruments Nanoscope IIIa controller running a Multimode AFM in contact mode both in air
and in aqueous solution. Cyclic voltammetry was
conducted with several different voltage ranges, in
deaerated solution, at 25 mV s-1.
3 RESULTS
3.1 Cytochrome imaging
Preliminary AFM imaging of c-type cytochromes
deposited on hematite in different ways are presented in Figure 1. In general, the results show that
c-type cytochromes adhere strongly to hematite surfaces, to the extent that AC AFM imaging modes are
not necessary in order to obtain images of cytochromes, even in aqueous solution. These results
imply that the forces of interaction between the cytochromes and the surface are probably greater than
the forces that hold the protein in a particular conformation, so that the molecules are likely denatured
upon sorption.
Figure 1. Top: (15x15 µm) Aggregated 10-20 nm particles of
cytochromes (lower left) deposited on a clean hematite surface
(upper right); Middle: 5x5 µm area cleared of cytochrome by a
high-force scan (~200 nN); steps and terraces are readily apparent; Bottom: 5x5 µm area after re-sorption of cytochrome
aggregates from the surrounding solution. The scan field near
center was created by scanning at high force, followed by subsequent imaging on about 2 nN net force.
Figure 2: Cyclic voltammograms of control solution containing buffer but no ferredoxin (Fdx) at neutral pH (thin solid
line), buffer solution with ferredoxin present at 0.5 mg L-1 at
neutral pH (finely dotted thin line), 0.5 mg L-1 ferredoxin acidified to pH 5.5 with HCl (finely dashed line), 0.5 mg L-1 ferredoxin acidified to pH 4.5 (coarsely dashed line), after addition
of ferredoxin solution to 0.88 mg L-1 (thick dotted line) with a
repeat scan (thick dashed line), and after a third addition to
ferredoxin solution to a concentration of 1.1 mg L-1.
3.2 Cyclic voltammetry, ferredoxin
Cyclic voltammetry of hematite electrodes in ferredoxin-free control buffer as well as ferredoxincontaining solutions are presented in Fig. 2. In the
initial control, cyclic voltammogram shows broad
hysteresis, as expected for the capacitance of a semiconductor. Scans started at the positive potential,
scanned toward the negative endpoint, and then returned to the positive end potential. The general increase in current toward negative potential is most
likely caused by incipient iron reduction at the
hematite surface, followed by its oxidation on the return to positive potential, similar to the observations
of Balko & Clarkson (2001). With the addition of
ferredoxin, a broad new oxidation peak appears in
the vicinity of 0 to –0.1 mV (there is a very broad
reduction peak at about –0.1 mV range, but it is obscured by the space-charge capacitance changes
caused by pH changes; both peaks are more evident
in the series of 5 scans presented in Fig. 3).
Acidification was undertaken in order to investigate whether creation of a positively charged surface
would attract the negatively charged redox functional region of the ferredoxin molecule (Wirtz et al.
2002). There are only slight increases in current in
response to lowered pH, however (Fig. 2). Addition
of another aliquot of ferredoxin (to 1.1 mg L-1) resulted in a dramatic change in the cyclic voltammo-
Figure 3: Sequence of 5 voltammograms showing slow increase in current associated with ferredoxin, implying its slow
sorption or reorientation. Solid thin line is scan 1; the reduction and oxidation peaks increase with each successive scan.
The last scan is shown by the thick solid line.
gram, with the oxidation peak shifting to about 0.2
mV (without ferredoxin, voltammograms to higher
positive potentials showed no oxidation peak). This
implies that either an increased surface coverage of
ferredoxin (perhaps together with other lipids and
proteins from the impure ferredoxin solution) results
in significant changes in the redox properties of the
ferredoxin molecule.
Returning to neutral pH and lower ferredoxin
concentrations with a new hematite sample, we investigated the possibility that sorption or ordering/orientation of ferredoxin on the hematite surface
is slow. Figure 3 shows increased redox current associated with ferredoxin on each of five successive
scans over a three minute period, implying slow
sorption or ordering/orientation kinetics. Addition
of cytochrome c (0.3 mg L-1 solution) to the solution
investigated in Figure 3 results in a drastically altered voltammogram (Fig. 4) in which, although the
oxidation peak has not shifted greatly, the reduction
peak occurs at about –0.3V. However, it is not
known whether this peak is due to ferredoxin or cytochrome. In any case, the redox properties of the
mixed impurity, ferredoxin, and cytochrome layer
on the hematite surface clearly change in response to
sorption of different substances to the surface, implying environment-dependent molecular properties.
These observations are similar, for example, to observed denaturing of cytochrome upon sorption to a
metal electrode, but stabilization of the cytochrome
by coadsorbing it with a lipid, bipyridine, or phosophate layer (Boussaad et al. 1998).
Figure 4: Cyclic voltammogram (4 successive scans) taken after addition of cytochrome c to the ferredoxin solution (see Fig.
3). First scan is the thin solid line and last scan is the thick
solid line with systematic changes in between.
4 CONCLUSION
Our results show that cytochrome c adheres strongly
to the hematite surface, allowing relatively easy nonAC AFM imaging in situ. The redox properties of
ferredoxin appear to depend on surface coverage as
well as the presence of other biomolecules. The results are consistent with the idea that the surface environment can affect the redox properties of sorbed
biomolecules through stabilization of particular conformations or molecular orientations. Future work
will concentrate on the role of ferredoxin as an electron shuttle between membrane-bound cytochrome
and a ferric oxide surface.
REFERENCES
Balko, B.A. & Clarkson, K.M. 2001. The effect of doping with
Ti(IV) and Sn(IV) on oxygen reduction at hematite electrodes. J. Electrochem. Soc. 148: 85-91.
Boussaad, S., Dziri, L., Arechabaleta, R., Tao, N.J. & Leblanc,
R.M. 1998. Electron transfer properties of cytochrome c
Langmuir-Blodgett films and interactions of cytochrome c
with lipids. Langmuir 14: 6215-6219.
Crofts, A.R. & Berry, E.A. 1998. Structure and function of the
cytochrome bc1 complex of mitochondria and photosynthetic bacteria. Curr. Opin. Struct. Biol. 8: 501-509.
Darrouzet, E., Valkova-Valchanova, M. & Daldal, F. 2002.
The [2Fe-2S] cluster Em as an indicator of the iron-sulfur
subunit position in the ubihydroquinone oxidation site of
the cytochrome bc1 complex. J. Biol. Chem. 277: 34643470.
Darrouzet, E. & Daldal, F. 2003. Protein-protein interactions
between cytochrome b and the Fe-S protein subunits during
QH2 oxidation and large-scale domain movement in the bc1
complex. Biochem. 42: 1499-1507.
Eggleston, C.M., Stack, A.G., Rosso, K.M., Higgins, S.R.,
Bice, A.M., Boese, S.W., Pribyl, R.D. & Nichols, J.J. 2003.
The structure of hematite (α-Fe2O3) (001) surfaces in aqueous media: Scanning tunneling microscopy and resonant
tunneling calculations of coexisting O and Fe terminations.
Geochim. Cosmochim. Acta 67: 985-1000.
Hernandez, M.E. & Newman, D.K. 2001. Extracellular electron transfer. CMLS Cell. Mol. Life Sci. 58: 1562-1571.
Kaufmann, F. & Lovley, D.R. 2001. Isolation and characterization of a soluble NADPH-dependent Fe(III) reductase from
Geobacter sulfurrreducens. J. Bacteriol. 183: 4468-4476.
Lovley, D.R., Coates, J.D., Blunt-Harris, E.L., Phillips, E.J.P.
& Woodward, J.C. 1996. Humic substances as electron acceptors for microbial respiration. Nature 382: 445-448.
Lovley, D.R. 2002. Dissimilatory metal reduction: From early
life to bioremediation. ASM News 68(5): 231-237.
Myers, C.R. & Myers, J.M. 1993. Role of menaquinone in the
reduction of fumarate, nitrate, iron(III) and manganese(IV)
by Shewanella putrefaciens MR-1. FEMS Microbiol. Lett.
114: 215-222.
Myers, C.R. & Myers, J.M. 1997. Cloning and sequence of
cymA, a gene encoding a tetraheme cytochrome c required
for reduction of iron(III), fumarate, and nitrate by Shewanella putrefaciens MR-1. J. Bacteriol. 179: 1143-1152.
Nevin, K.P. & Lovley, D.R. 2000. Lack of production of electron-shuttling compounds or solubilization of Fe(III) during
reduction of insoluble Fe(III) oxide by Geobacter metallireducens. Appl. Environ. Microbiol. 66(5): 2248-2251.
Newman, D.K. & Kolter, R. 2000. A role for excreted quinones
in extracellular electron transfer. Nature 405: 94-97.
Turick, C.E., Tisa, L.S. & Caccavo, F. Jr. 2002. Melanin production and use as a soluble electron shuttle for Fe(III) oxide reduction and as a terminal electron acceptor by Shewanella algae BrY. Appl. Environ. Microbiol. 68(5): 24362444.
Wirtz, M., Klucik, J. &Rivera, M. 2000. Ferredoxin-mediated
electrocatalytic dehalogenation of haloalkanes by cytochrome P450cam. J. Am. Chem. Soc. 122: 1047-1056
Zachara, J.M., Frederickson, J.K., Li, S.-M., Kennedy, D.W.,
Smith, S.C. & Gassman, P.L. 1998. Bacterial reduction of
crystalline Fe3+ oxides in single phase suspensions and subsurface materials. Am. Mineral. 83: 1426-143.