High-Affinity Binding and Direct Electron Transfer to Solid Metals by theShewanella oneidensis MR-1 Outer Membrane c-type Cytochrome OmcA

Yijia Xiong,† Liang Shi,† Baowei Chen,† M. Uljana Mayer,† Brian H. Lower,‡ Yuri Londer,‡Saumyaditya Bose,‡ Michael F. Hochella,‡ James K. Fredrickson,†,‡ and Thomas C. Squier*,†

DiVision of Biological Sciences and EnVironmental Molecular Science Laboratory,Biogeochemisty Grand Challenge Program, Pacific Northwest National Laboratory, Richland, Washington 99352

Received May 19, 2006; E-mail: thomas.squier@pnl.gov

The mechanisms by which bacterial outer membrane-associatedelectron transport proteins interact with extracellular electron accep-tors, including Fe and Mn oxides, is poorly understood. The 85 kDaouter membrane decaheme cytochrome OmcA (SO1779) from thedissimilatory metal-reducing bacterium (DMRB)Shewanella oneiden-sisMR-1 can reduce soluble Fe(III) and other metal chelates,1 andhas previously been suggested to function in concert with othermembrane proteins as one of the terminal electron donors in themetal reductase protein complex ofShewanella oneidensisMR-1.2

Shewanella’smetabolic diversity has considerable promise for thebioremediation of metal and radionuclide contaminants as well asin the design of microbial fuel cells.3 Biofuel cells offer a potentialmeans to couple the breakdown of biowastes to generate electricalcurrent.4 Miniaturization of these fuel cells is dependent on the elim-ination of the membrane between the cathode and anode compart-ments.5 It has been demonstrated that the immobilization of redox-active proteins on electrodes renders this membrane unnecessary.6

The identification of a purified metal-reducing enzyme able to den-sely bind and directly donate electrons to commonly used iron-oxide-coated electrodes7 has great potential to contribute to fuel cell design.This will also contribute to understanding how DMRB transferelectrons to solid metal oxides in the environment, which mayinvolve direct interactions with membrane proteins or an indirectmechanism through mineral solubilization or electron shuttling. Toidentify the terminal electron donors in theS. oneidensismetalreductase system and to explore whether isolated proteins candirectly bind and mediate electron-transfer reactions to reduce solidmetals, we have purified OmcA and measured its ability to bindand transfer electrons to solid Fe2O3 in the mineral hematite.

Hematite nanoparticles were generated using the forced hydroly-sis of Fe3+(OH2)6 ions to form particles,8 whose average radiuswas measured to be 11( 2 nm at pH 5 (see SupportingInformation). At the physiological pH for the catalytic activity ofOmcA (pH 7.5), hematite self-associates to form a broad distributionof size classes ranging from about 10 nm (=1 particle) to largeaggregates with an apparent size of about 400 nm [see SupportingInformation (SI) Figure S1]. OmcA directly associates with addedhematite, as evidenced by the co-sedimentation of approximately40% of the OmcA in solution with hematite particles uponcentrifugation (Figure 1A), permitting a determination of the bindingaffinity through the measurement of the concentration of OmcAbound to hematite relative to that in water (i.e., the partitioncoefficient). We find that approximately 0.2 mg of OmcA bindsper mg of hematite (i.e., 2.5 nmol OmcA per mg of hematite). Fromthe average radius of hematite at pH 7.5 (i.e., 400 nm), ap-proximately 106 OmcA proteins bind to each particle (i.e., 1014

OmcA proteins bind per cm2), corresponding to a partition

coefficient of approximately 2× 105 (∆Go′ ) -28 kJ/mol). Asimilar high-affinity binding interaction is measured using decreasesin the intrinsic tryptophan fluorescence of OmcA to assess bindingas a function of added hematite. Furthermore, upon association withhematite there is a large decrease in the solvent accessibility of thetryptophans in OmcA to a soluble acrylamide quencher (Figure 1B),consistent with a direct binding interaction between OmcA andhematite. In contrast, there is no significant binding between OmcAand other minerals or particles (SI Figures S3 and S4), indicatingspecific binding between OmcA and hematite.

In association with hematite, OmcA is catalytically active:oxidation of protein hemes, as measured from time-dependentchanges in theR Soret absorption peak at 552 nm,2 directly trackswith protein binding to hematite under anoxic conditions with amaximal activity of about 60 nmol mg-1 OmcA min-1. Since OmcAcan be directly reduced by NADH and other metabolic cofactors,2

the high-affinity interaction between OmcA and hematite providesa means to couple the generation of reducing power to an electrodesurface. To confirm the binding specificity, and to detect possiblechanges in the internal dynamics of OmcA upon association withhematite, we labeled OmcA with the chromophore Alexa-488 andhave used fluorescence correlation spectroscopy (FCS) to measurefluorescence intensity fluctuations associated with both changes ininternal motion and decreases in the translational diffusion of OmcAupon association with hematite.9 These measurements utilized aconfocal microscope using a pair of SPCM-AQR-14 avalanchephotodiodes to perform pseudocross-correlation measurements thatefficiently remove after-pulse effects that correspond to spontaneoussignals associated with individual photodetectors that can broadenand distort fluorescence intensity traces. The fluorescence intensitytrace for Alexa488-labeled OmcA in the absence of hematite ischaracterized by a high density of low-amplitude bursts, whosefrequency is indicative of the concentration of protein in solution(Figures 2A and S5). Upon addition of hematite, there is a dramaticincrease in the amplitude (brightness) of some fluorescence burststhat is indicative of multiple OmcA proteins binding to hematite

† Division of Biological Sciences.‡ Environmental Molecular Science Laboratory.

Figure 1. Functional association between 1.0µM OmcA and hematitenanoparticles detected by (A) electron-transfer activity (]) or direct bindingmeasured by loss of OmcA through co-sedimentation with hematite particles(O) following centrifugation (16000g for 10 min) or diminished intrinsicfluorescence (b) and (B) decreased solvent accessibility (F0/F) of Trp inOmcA following hematite addition (30µg/mL), where F0/F is thefluorescence change upon acrylamide addition.

Published on Web 10/11/2006

13978 9 J. AM. CHEM. SOC. 2006 , 128, 13978-13979 10.1021/ja063526d CCC: $33.50 © 2006 American Chemical Society

particles (Figure 2B). Other heme-containing proteins do not bindto hematite (SI Figure S6). A quantitative consideration of thesample heterogeneity is possible from a consideration of thefluorescence correlation curves associated with the fluorescenceintensity traces (Figure 2, C and D). The correlation curve isdescribed by the equation:

N is the number of molecules in the focus point,S is the ratio ofhalf-height to radius (ω) of the focus point,τ is the lag time betweenfluorescence bursts,τD is the apparent diffusion time of the mole-cule, andDt is the translational diffusion coefficient. The radius ofthe focal point (ω) was measured to be 270 nm using both the freedye Alexa488 and a standard fluorescently labeled bead with aknown radius (r ) 50.5 nm), whose measured translational diffusiontime has a narrow distribution centered at 4.73 ms, correspondingto a calculated radius of 52 nm (see SI Figure S2). However, thesize of heterogeneous samples cannot be accurately determined byassuming a homogeneous monodisperse solution. Rather, a distribu-tion of diffusion times is described by the following function:

whereG(τ) is the FCS curve with diffusion timeτi andAi is thefractional amplitude ofG(τ). Fits to the correlation function involvedmodification of the Matlab program Smcontinbot.10 The small andrandom residuals indicate that the resulting distribution of diffusiontimes accurately describe the sample heterogeneity (Figure 2, Eand F).

The translational motion of OmcA in the absence of hematite isdescribed by a multimodal distribution of diffusion times centerednear 160µs, 800µs, and 20 ms (Figure 2G). Using the Stokes-Einstein equation to calculate the hydrodynamic radius (see SIFigure S2), the intermediate diffusion time (i.e.,τD ) 800µs) withan apparent radius of about 9 nm is consistent with the size ofOmcA, which can form a dimer in solution, while the largerdiffusion time (i.e.,τD ) 20 ms) corresponds to a higher-orderoligomer.2 The smaller diffusion time centered near 160µs isassigned to internal domain motions that function to modulate the

fluorescence intensity of Alexa488. In the presence of hematite thetranslational diffusion time for the OmcA dimer is shifted towarda larger time constant (i.e., 1.2 ms), consistent with OmcAmolecules binding to small hematite particles (Figure 2H); inaddition a larger diffusion time centered near 800 ms is observedthat corresponds to the size of hematite particles measured bydynamic light scattering and is associated with approximately 106

OmcA proteins bound per particle (Figure 1A). Likewise, uponOmcA binding to hematite there is a substantial increase in therelative area associated with OmcA domain motions, whichincreases from 50 to 70% of the integrated peak area (Figure 2, Gand H). Thus, binding to hematite increases domain motions thatmay facilitate electron-transfer reactions between heme clusters inOmcA by modulating their spatial arrangement.11

In summary, we have shown that purified OmcA binds anddensely covers the surface of hematite (approximately 1014 OmcAproteins bind per cm2) and reduces Fe(III) with a maximal velocityof approximately 60 nmol/mg/min, which corresponds to an electronflux of about 1013 electrons /cm2/s that approaches observed fluxesin the most efficient bioreactors.3

Acknowledgment. Supported by Genomics:GTL Program ofthe Department of Energy (DOE- OBER) and an EMSL ScientificGrand Challenge Project at PNNL, which is operated for DOE byBattelle under Contract DE-AC05-76RL0 1830.

Supporting Information Available: Procedures and characteriza-tion data; complete refs 2a, 2b, 2c. This material is available free ofcharge via the Internet at http://pubs.acs.org.

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(2) (a) Shi, L.; et al.J. Bacteriol.2006, 188, 4705-4714. (b) Gorby, Y. A.;et al.Proc. Natl. Acad. Sci. U.S.A.2006, 103, 11358-11363. (c) Marshall,M. J.; et al.PLoS Biol.2006, 4, 1324-1333. (d) Wigginton, N. S.; Rosso,K. M.; Lower, B. H.; Shi, L.; Hochella, M. F., Jr.Geochim. Cosmochim.Acta 2006. In press.

(3) (a) Viamajala, S.; Peyton, B. M.; Apel, W. A.; Petersen, J. N.,Biotechnol.Bioeng.2002, 78, 770-778. (b) Ringeisen, B. R.; Henderson, E.; Wu, P.K.; Pietron, J.; Ray, R.; Little, B.; Biffinger, J. C.; Jones-Meehan, J. M.EnViron. Sci. Technol.2006, 40, 2629-2634.

(4) (a) Bond, D. R.; Lovley, D. R.Appl. EnViron. Microbiol. 2003, 69, 1548-1555. (b) Kim, B. H.; Park, H. S.; Kim, H. J.; Kim, G. T.; Chang, I. S.;Lee, J.; Phung, N. T.Appl. Microbiol. Biotechnol.2004, 63, 672-681.(c) Liu, H.; Ramnarayanan, R.; Logan, B. E.EnViron. Sci. Technol.2004,38, 2281-2285.

(5) Kim, H.-H.; Mano, N.; Zhang, Y.; Heller, A.J. Electrochem. Soc.2003,150, A209-A213.

(6) (a) Chen, T.; Barton, S. C.; Binyamin, G.; Gao, Z.; Zhang, Y.; Kim, H.H.; Heller, A. J. Am. Chem. Soc.2001, 123, 8630-8631. (b) Mano, N.;Kim, H. H.; Zhang, Y.; Heller, A.J. Am. Chem. Soc.2002, 124, 6480-6486. (c) Mano, N.; Mao, F.; Heller, A.J. Am. Chem. Soc.2002, 124,12962-12963. (d) Mano, N.; Mao, F.; Heller, A.J. Am. Chem. Soc.2003,125, 6588-6594. (e) Mano, N.; Mao, F.; Heller, A.ChemBioChem2004,5, 1703-1705. (f) Mano, N.; Mao, F.; Shin, W.; Chen, T.; Heller, A.Chem. Commun. (Camb) 2003, 518-519. (g) Soukharev, V.; Mano, N.;Heller, A. J. Am. Chem. Soc.2004, 126, 8368-8369.

(7) Kim, J. R.; Min, B.; Logan, B. E.Appl. Microbiol. Biotechnol.2005, 68,23-30.

(8) Madden, A. S.; Hochella, M. F., Jr.; Michael, F.Geochim. Cosmochim.Acta 2005, 69, 389-398.

(9) (a) Chattopadhyay, K.; Saffarian, S.; Elson, E. L.; Frieden, C.Proc. Natl.Acad. Sci. U.S.A.2002, 99, 14171-14176. (b) Haupts, U.; Maiti, S.;Schwille, P.; Webb, W. W.Proc. Natl. Acad. Sci. U.S.A.1998, 95, 13573-13578. (c) Miguel AÄ ngel Medina, P. S.BioEssays2002, 24, 758-764.(d) R. Rigler, E. S. E.Fluorescence correlation spectroscopy, theory andapplication; Springer: New York, 2001. (e) Whittier, J. E.; Xiong, Y.;Rechsteiner, M. C.; Squier, T. C.J. Biol. Chem.2004, 279, 46135-46142.

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JA063526D

Figure 2. Fluorescence intensity traces (A, B) and correlation curves withfits (red) (C, D) for 10 nM Alexa488-labeled OmcA in the absence (leftpanels) or presence (right panels) of hematite (16 ug/mL). Residuals fromfits to the correlation data (E, F) and distribution of diffusion times (G, H).

G(τ) ) 1N

1

(1 + ττD

)‚x1 + τS2τD

where τD ) ω2

4Dt

f(τ) ) ∑i

AiG(τi)

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J. AM. CHEM. SOC. 9 VOL. 128, NO. 43, 2006 13979

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Supplementary Material High-Affinity Binding and Direct Electron Transfer to Solid Metals by Purified Metal

Reducing Protein OmcA Decaheme Cytochrome. Yijia Xiong1, Liang Shi1, Baowei Chen1, M. Uljana Mayer1, Brian H Lower2, Yuri Londer2,

Saumyaditya Bose2, Michael F. Hochella2, James K. Fredrickson1,2, and Thomas C. Squier1

1Division of Biological Sciences and 2Environmental Molecular Science Laboratory Biogeochemisty Grand Challenge Program; Pacific Northwest National Laboratory, Richland, WA 99352

RECEIVED DATE (automatically inserted by publisher); thomas.squier@pnl.gov

Institutional Affiliations of Biogeochemistry Grand Challenge Team: Saumyaditya Bose and Michael F. Hochella: Department of Geosciences, Virginia Polytechnical Institute, Blacksburg, VA 24061. Yuri Y. Londer: Biosciences Division, Argonne National Laboratory, 9700 South Cass Avenue, Argonne, IL 60439. Brian H. Lower* and James K. Fredrickson+: Chemical* or Biological+ Sciences Divisions, Fundamental Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA 99352. OmcA and Experimental Buffer: Wild-type recombinant OmcA was expressed with V5 and His6 tags at the C-terminus, and was purified using immobilized metal affinity columns (IMAC).2a No additional tags (e.g., tetracysteine tags) were employed in these measurements. OmcA ( 85

g/mL or 1.0 M) in 20 mM HEPES (pH 7.5), 0.15 M NaCl, and 8 mM CHAPS (CMC = 8-10 mM) was used in all experiments. In all cases, hematite was preincubated in 20 mM HEPES (pH 7.5), 0.15 M NaCl, and 8 mM CHAPS and the detergent concentration was kept constant. Similar OmcA binding to hematite was observed when the lipid binding site near the N-terminus of the protein was truncated (in the absence of added detergent), ensuring that binding to hematite was specifically associated with the functional properties of OmcA and its ability to transfer electrons directly to hematite. Hematite Synthesis: Hematite nanoparticles were synthesized as previously described.8 Briefly, this involved slowly dripping 60 mL of 1 M ferric nitrate solution into 750 ml of boiling ultrafiltered and doubly distilled MilliQ water. After the drip solution was consumed, the nanoparticle solution was removed from the heat and the synthesis suspension was cooled overnight. The nanoparticle solution was then dialyzed in high grade regenerated cellulose tubular membranes (Cellu Sep H1, 6000-8000 molecular weight cut-off) against doubly distilled MilliQ water until the conductivity of the dialysis water reached that of pure MilliQ. The suspensions were then poured into HDPE bottles for storage. Some of the suspension was freeze dried in a Labconco FreeZoen freeze dry system prior to characterization. Hematite Characterization: Mineralogical characterization was carried out by powder X-ray diffraction (XRD) on a Philips X’Pert MPD system with a Cu anode operating at a wavelength of 1.5406Å (CuKα1) as the radiation source (see below). Specimens were prepared by grinding the samples in an agate mortar and pestle to fine powder and then placed on off axis quartz plates (18mm dia x 0.5 mm DP cavity). Diffraction patterns were recorded with a proportional counter detector over a 10-60° two theta scan range at a rate of 0.025°/sec. The X-ray diffraction powder pattern revealed the synthetic samples to be pure hematite with no detectable amounts of impurities.

0

100

200

300

400

500

600

700

Inte

nsity

(Cou

nts)

033-0664> Hematite - Fe 2O3

20 30 40 502Theta°

[h60111b.dif] XRPD: Sample #10 (Test 01)

Powder X-ray diffraction pattern (left) and transmission electron micrograph (right) of hematite nanoparticles.

2

Particle morphology, size distribution, and electron diffraction patterns were obtained by transmission electron microscopy (TEM) (see above). Specimens were prepared by placing a drop of hematite suspension onto a 200 mesh formvar-coated copper grid (stabilized with evaporated carbon film) and allowed to evaporate. The products were observed in a Phillips EM 420T Scanning Transmission Electron Microscope operated in bright field mode at 100 KeV. Size distribution and morphology was estimated by observing approximately 90-100 particles from TEM negatives on a lightbox with a 10x magnifier with measuring scale. The TEM micrographs show aggregated pseudo-hexagonal platelets. Electron diffraction rings indicate hematite. The mean particle size and standard deviation as determined from a normal fit to the size distribution histogram was 11 ± 2 nm. Specific surface area was determined by nitrogen gas adsorption by the Brunauer-Emmett-Teller (BET) method. The freeze-dried powder was degassed overnight at 100 °C followed by a 6-point BET isotherm in a Quantachrome Nova 1000 N2 adsorption analyzer with N2 as the adsorbate. The BET specific surface area was determined to be 95.95 (m2/g). Solubility of Hematite: The solubility of solid minerals is determined by the free energy of dissolution, which in general is very low for Fe(III) oxides (i.e., in the nanomolar range in the pH range between 4-10 in the absence of complexing agents). Indeed, minimum solubility is observed between pH 7 and pH 8; around pH 7.5 the solubility of hematite (the least soluble iron oxide) is approximately 5 pM independent of added salts12. Solubilities are enhanced for small particles, such that the calculated solubility of Fe(III) increases by about two order of magnitude upon decreasing the particle size to about 10 nm. However, even under these latter conditions the concentration of free Fe(III) in solution is substantially less than 1 nM. Thus, the concentration of free (unchelated) iron is less than 0.1% of the concentration of OmcA used in enzyme activity measurements (i.e., 1 uM), where electron transfer rates were 60 nmol/mg/min in the presence of hematite (Figure 1A). In comparison, prior activity measurements using soluble iron were made in the presence of 10 mM Fe(III)-NTA, and no measurable activity was detected in the absence of NTA2a. Reductase Activity of OmcA: The reductase activity of OmcA toward hematite was assayed using a variation of a previously described procedures.1,13-15 Briefly, OmcA was chemically reduced by sodium dithionite. The reaction was carried out in anaerobic glass cells (STARNA, Atascadero, CA, USA), that contained purified OmcA (1 µM) in 1 mL of 20 mM HEPES (pH 7.5), 150 mM NaCl, 0.5% CHAPS, 10% glycerol and different concentrations of hematite, rotated in a Labquake tube rotator (Fisher, Pittsburg, PA, USA). The absorption spectrums, OD540-560, of OmcA were recorded by an Agilent 8453 UV/Vis diode array spectrometer (Palo Alto, CA, USA) in an anaerobic chamber to measure the oxidation rate of OmcA by hematite. Controls included omission of hematite. All solutions were purged with 80% N2/20% CO2 (room temperature, 2 hr) before use, and all reactions were performed at room temperature in an anaerobic chamber with O2 level of 1 ppm. After the initial rates (less than 10% of the hemes were oxidized) were calculated, they were fitted to the Michaelis-Menten equation to yield km and kcat by using OriginPro7.5 software. Covalent Attachment of Alexa-488 to OmcA: OmcA was fluorescently labeled using amine reactive Alexa Fluor® 488 carboxylic acid succinimidyl ester (cat #A20000, Invitrogen, Eugene, OR) at a molar stoichiometry of about 2:1 (dye to protein). Briefly, Alexa488 (40 M)was incubated with OmcA [0.6 mg/ml in 10 mM Na2PO4 (pH 7.5), 150 mM NaCl, 0.5% CHAPS] for two hours at room temperature, and Alexa488-labeled OmcA was separated from free dye using size exclusion chromatography (i.e., a Sephedex G25 column) into 20 mM HEPES (pH 7.5), 150 mM NaCl and 0.5% CHAPS. The concentration of Alexa488 bound to OmcA was determined by its absorbance at 494 nm ( ε494nm = 73000 M-1 cm-1).

Purification of SO0717: The predicted mature fragment of SO0717 was cloned as a C-terminal fusion to a hybrid 6 × His-Maltose binding protein affinity tag followed by a cleavage site for TEV protease.16 The plasmid was transformed into E. coli strain BL21(DE3) harboring auxiliary plasmid pEC86 that contains genes for cytochrome c maturation.17 E. coli cultures were grown aerobically to mid-exponential phase at 30°C and 250 rpm and induced with 20 µM IPTG. The incubation continued overnight at 30 °C and 200 rpm. Next morning the cells were harvested and resuspended in 100 mM Tris-HCl (pH 8.0), 20% sucrose, and 0.5 mM EDTA containing 0.5 mg/ml lysozyme and protease inhibitor cocktail for bacterial cells (Sigma). 30 ml of the buffer was used to resuspend a pellet from 1 L of culture. Resuspended cell were incubated at room temperature for 15 min. Then an equal volume of ice-cold 1 M NaCl solution was added and the cells were incubated on ice with gentle shaking for 15 min and centrifuged at 12,000 × g for 20 min at 4°C. The supernatant constituted the periplasmic fraction. Then imidazole and MgCl2 were added to final concentrations of 5 mM and 2 mM, respectively, and the prep was purified on a 20 mL Ni Sepharose 6 Fast Flow (GE Healthcare) column equilibrated with buffer A [20 mM Tris-HCl (pH 8.0), 0.5 M NaCl, and 5 mM imidazole] according to the manufacturer’s instructions. Then TEV protease was added in 1:20 (w/w) ratio and the sample was dialyzed overnight against buffer A. The dialyzed sample was loaded onto the same re-equilibrated Ni Sepharose 6 Fast Flow column and the flow-through was collected and dialyzed against 20 mM HEPES (pH 7.5) and 150 mM NaCl. The purified protein was frozen at – 80 C prior to use.

3

ADDITIONAL REFERENCES: Complete References for those Abbreviated in the Manuscript: 2a. Shi, L.; Chen, B.; Wang, Z.; Elias, D. A.; Mayer, M. U.; Gorby, Y. A.; Ni, S.; Lower, B. H.; Kennedy, D. W.; Wunschel, D. S.; Mottaz, H. M.; Marshall, M. J.; Hill, E. A.; Beliaev, A. S.; Zachara, J. M.; Fredrickson, J. K.; Squier, T. C., Isolation of high-affinity functional protein complex between OmcA and MtrC: Two outer membrane decaheme c-type cytochromes of Shewanella oneidensis MR-1. J Bacteriology 2006, 188, 4705-4714. 2b. Gorby, Y. A.; Yanina, S.; McLean, J. S.; Rosso, K. M.; Moyles, D.; Dohnalkova, A.; Beveridge, T. J.; Chang, I. S.; Kim, B. H.; Kim, K. S.; Culley, D. E.; Reed, S. B.; Romine, M. F.; Saffarini, D. A.; Hill, E. A.; Shi, L.; Elias, D. A.; Kennedy, D. W.; Pinchuk, G.; Watanabe, K.; Ishii, S.; Logan, B.; Nealson, K. H.; Fredrickson, J. K., Electrically conductive bacterial nanowires produced by Shewanella oneidensis strain MR-1 and other microorganisms. Proc Natl Acad Sci U S A 2006, 103, (30), 11358-11363. 2c. Marshall, M. J.; Beliaev, A. S.; Dohnalkova, A. C.; Kennedy, D. W.; Shi, L.; Wang, Z.; Boyanov, M. I.; Lai, B.; Kemner, K. M.; McLean, J. S.; Reed, S. B.; Culley, D. E.; Bailey, V. L.; Simonson, C. J.; Saffarini, D. A.; Romine, M. F.; Zachara, J. M.; Fredrickson, J. K., c-Type Cytochrome-Dependent Formation of U(IV) Nanoparticles by Shewanella oneidensis. PLoS Biol 2006, 4, 1324-1333. Additional References Not Already Cited: 12. Cornell, R.; Schwertmann, U., The Iron Oxides: Structure, Properties, Reactions, Occurances and Uses. WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.: 2003; p pp. 201-220. 13. Field, S. J.; Dobbin, P. S.; Cheesman, M. R.; Watmough, N. J.; Thomson, A. J.; Richardson, D. J., Purification and magneto-optical spectroscopic characterization of cytoplasmic membrane and outer membrane multiheme c-type cytochromes from Shewanella frigidimarina NCIMB400. J Biol Chem 2000, 275, 8515-8522. 14. Magnuson, T. S.; Hodges-Myerson, A. L.; Lovley, D. R., Characterization of a membrane-bound NADH-dependent Fe(3+) reductase from the dissimilatory Fe(3+)-reducing bacterium Geobacter sulfurreducens. FEMS Microbiol Lett 2000, 185, 205-211. 15. Pitts, K. E.; Dobbin, P. S.; Reyes-Ramirez, F.; Thomson, A. J.; Richardson, D. J.; Seward, H. E., Characterization of the Shewanella oneidensis MR-1 decaheme cytochrome MtrA: expression in Escherichia coli confers the ability to reduce soluble Fe(III) chelates. J Biol Chem 2003, 278, 27758-27765. 16. Donnelly, M. I.; Zhou, M.; Millard, C.S.; Clancy, S.; Stols, L., Eschenfeldt WH, Collart FR, Joachimiak A. An expression vector tailored for large-scale, high-throughput purification of recombinant proteins. Protein Expr Purif. 2006, 47, 446-454. 17. Arslan, E.; Schulz, H.; Zufferey, R.; Kunzler, P.; Thöny-Meyer, L., Overproduction of the Bradyrhizobium japonicum c-type cytochrome subunits of the cbb3 oxidase in Escherichia coli. Biochem Biophys Res Commun 1998, 251, 744–747. 18. Stipp, S. L.; Hochella, M. F., Jr., Structure and bonding environments at the calcite surface as observed with X-ray photoelectron spectroscopy (XPS) and low energy electron diffraction (LEED). Geochimica et Cosmochimica Acta 1991, 55, 1723-1736. 19. Lower, S.K., Hochella, M.F., Jr. and Beveridge, T.J. Bacterial recognition of mineral surfaces: nanoscale interactions between Shewanella and alpha-FeOOH. Science 2001, 292, 1360-1363.

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Figure S1. Average spherical radius determined from fits (red lines) to dynamic light scattering (DLS) data (circles) for (A) hematite in 20 mM HEPES (pH 7.5), 150 mM NaCl, and 0.5% w/v CHAPS in comparison to data collected for polystyrene bead standards whose radii are known to be 950 nm (Fluosphere cat #F8887, Molecular Probes, Eugene OR) (B) or 545 nm (Polystyrene bead cat #16905, Polysciences Inc, Warrington PA) (C). The normalized experimental curve was fit to the following: G(t) = A × exp[-2Dtq2t], where A is an instrumental coefficient, Dt is the translational diffusion coefficient, and q is the scattering vector ]/)2/(sinn4[ λθπ , where n is the refract index, θ is the scattering angle and λ is the wavelength of the scattering

light (λex = 633 nm). The apparent radius (r) for a spherical particle was calculated from the Stokes-Einstein equation tD6/kTr πη= , where r is the

hydrodynamic radius of the molecule, k is the Boltzmann constant ( 1.38×10-23 J/K), T is the absolute temperature (293 ºK), and η is the viscosity of water (0.001 kg/m/s, or 0.01 poise). From the data the average radius of hematite was approximately 400 nm (A), while that of the polystyrene beads were respectively determined to be 950 nm (B) and 546 nm (C).

10-3 10-2 10-1

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0.010

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0.010

0.015

0.000

0.005

0.010

0.015

polystyrene bead (r = 546 nm)C

Lag Time (sec)

polystyrene bead (r = 950 nm)

G(τ

)B

hematite ( r = 400 nm)

A

5

Figure S2. Fluorescence intensity traces (A) and derived experimental correlation curve (black) and fit (red) (B), residuals of the fitting (C), and the distribution of diffusion times (τD) (D) calculated using an aqueous suspension of standard beads with a known radius of 50.5 nm that was labeled with a red fluorophore (FluoSpheres F8887, 580/605, Molecular Probes Inc. Eugene OR). The translational diffusion coefficient (Dt) calculated from:

D

2

t 4D

τ×ω

= , where for τD = 4.4 × 10-3 s one can calculate that Dt = 4.1 × 10-8 cm2 / s.

From the Stokes-Einstein equation the radius (r) of a spherical particle can be calculated according to:

10-5 10-4 10-3 10-2 10-1 100 101

-0.04

0.00

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3

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1

2

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4

0.0

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D

Frac

tion

Diffusion Time (s)

C

Res

idua

l

Lag Time (sec)

B

G(τ

)

10010-5 10-310-4 10110-1

A

Pho

ton

Cou

nts

Time (s)

10-2

nm52)seccm101.4()cmsecdyne01.0(6

)K293()degcmdyne1038.1(D6

kTr 1282

116

t=

×π°×

=ηπ

= −−−

−−

6

Figure S3. Loss of OmcA through co-sedimentation with hematite (α-Fe2O3), goethite (α-FeOOH) and diaspore (α-AlOOH) particles following centrifugation (16,000 x g for 10 min) following addition of 0.16 mg/mL hematite or 1.6 mg/mL goethite or diaspore. Buffer is 20 mM HEPES (pH 7.5), 150 mM NaCl, and 0.5% w/v CHAPS. Natural samples of goethite and diaspore were obtained from the Department of Geosciences Museum at Virginia Tech. Each sample was crushed and wet sieved through a 45 m sieve. Those particles passing through the filter were collected and cleaned of adventitious carbon18, and confirmed to be solely composed of either goethite or diaspore by X-ray diffraction analysis. Our results, demonstrating a functional and high-affinity interaction between OmcA and hematite suggests a direct role for this metal reductase in mediating electron transfer to hematite. The lack of binding of purified OmcA to goethite is in contrast to prior measurements using living Shewanella that demonstrated a high affinity interaction with goethite under anaerobic conditions when metal reductases are highly expressed in the outer membranes of Shewanella19. However, no prior measurements considered the ability of a purified metal reductase to bind and transfer electrons to solid minerals, and the nature of the bacterial-mineral interaction remains unclear and could involve nonspecific interactions involving, for example, polysaccharides. The inability of OmcA to bind to goethite is consistent with our prior suggestions that functional binding interactions between multiple metal reductases (e.g., OmcA and MtrC) function to enhance binding affinities between Shewanella and different minerals.2a Future measurements will endeavor to document the binding interactions between different metal reductases and the range of minerals in soils that are used as electron transfer acceptors in the colonization of soils by Shewanella and other metal reducing bacteria.

Hematite Goethite Diaspore0.0

0.5

1.0

1.5

2.0

2.5

3.0

Om

cAbi

ndin

g (nm

ol/m

g)

7

Figure S4. Fluorescence intensity traces for 100 nM Alexa488-labeled OmcA in the absence (A) or presence (B) of silica microspheres (100 M) (R = 500 nm; Polysciences Inc., Cat # 24323) and their fluorescence correlation spectroscopy curves(C), respectively. Buffer is 20 mM HEPES (pH 7.5), 150 mM NaCl, and 0.5% w/v CHAPS.

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110-110-210-310-410-510-6

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OmcA OmcA + Silica Particle

(R = 500 nm)

G(τ

)Lag Time (sec)

Nor

mal

ized

Pho

ton

Cou

nts

Time (s)

B

A

8

Figure S5. Fluorescence intensity trace of 10 nM (A), 50 nM (B) and 100 nM (C) Alexa488-labeled OmcA in 20 mM HEPES (pH 7.5), 150 mM NaCl, and 0.5% w/v CHAPS. In all cases, the sample time was 100 s.

0.0 0.1 0.20246

0.0 0.1 0.20246 0.0 0.1 0.20246

A

C

Time (s)

Fl

uore

scen

ce In

tens

ity

B

9

Figure S6. Fluorescence intensity traces for 100 nM Alexa488-labeled SO0717 in the absence (A) or presence (B) of hematite (16 g/ml) and their fluorescence correlation spectroscopy curves (C), respectively. Buffer is 20 mM HEPES (pH 7.5) and 150 mM NaCl. SO0717 is a cytochromes containing protein isolated from Shewanella oneidensis.

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8C

SO0717 SO0717 + Hematite

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)

Lag Time (sec)110-1 10-210-310-410-510-6

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mal

ized

Pho

ton

Cou

nts

Time (s)

A