Thrust area 4: How redox proteins on the exterior of the outer membrane of Shewanella accomplish interfacial electron transfer to the Fe(III) oxide surface.
EMSL Project ID
13890
Abstract
A multidisciplinary team of scientists is investigating the mechanisms of electron transfer from metal-reducing bacteria to Fe(III) oxide surfaces as part of a PNNL/DOE-BER Grand Challenge in Biogeochemistry (BGC) being led by Drs. Zachara and Fredrickson of PNNL. Dr. Carrick Eggleston (CME) is a leading expert on the molecular surface and electronic properties of Fe(III) oxides and their experimental interrogation by scanning probe microscopy and optical spectroscopy. Dr. Egglestons' recent research on redox protein interactions with hematite distinguish him as one of a few scientists internationally to address this exceedingly complex experimental system that is the scientific core of the BGC. Dr. Eggleston was a participant in the two BGC planning workshops (e.g., Nov. 2003 and Sept. 2004) and his continued participation and research efforts in the BGC are required to understand how redox proteins on the exterior of the outer membrane of Shewanella accomplish interfacial electron transfer to the Fe(III) oxide surface. The success of the BGC requires that the following tasks be conducted at the University of Wyoming under the direction of Dr. Eggleston. 1) Characterization of Fe(III) oxide surfaces in the absence and presence of redox proteins.
Dr. Eggleston's group will perform scanning tunneling and atomic force microscopy (STM and AFM; together, scanning probe microscopy) on Fe(III) oxide surfaces both before and after contact with redox proteins to be provided by PNNL, or after contact with various Shewanella species and mutants (as part of other BGC member research). Dr. Eggleston's group will also perform ICP-MS analyses of oxide mineral digests to establish impurity (and thus charge carrier) content. Dr. Eggleston will collaborate with BGC participants at PNNL, Stanford University, Virginia Tech University, and the University of Montana to identify and exchange a series of common MBE, synthetic, and natural hematite and magnetite samples for collaborative research. Dr. Eggleston will assist the BGC research group in identifying suitably doped synthetic and natural hematites for collaborative protein-mineral and whole cell-mineral studies that are amenable to STM study.
2) Characterization of redox metalloproteins (mtrA, mtrB, and mtrC) and their interactions with Fe(III) oxide surfaces.
Dr. Eggleston's group will perform wet-chemical sorption, UV-visible spectroscopy, SPM imaging, and cyclic and differential-pulse voltammetry measurements for purified Shewanella cytochromes to be provided by PNNL as a basis for understanding the physicochemical interactions of redox proteins at the Fe(III) oxide-water interface. Dr. Eggleston will coordinate his characterization measurements carefully with Dr. David Richardson at the University of East Angelia, who will also be performing electrochemical measurements on the purified cytochromes. Electrochemical characterization of single molecule cytochromes provided by PNNL, possibly isolated in membrane fragments or liposomes, should be considered using electrochemical scanning tunneling microscopy (although this work should be considered high-risk but also high-reward given its many difficulties).
Dr. Eggleston's group will evaluate the electrochemical properties of mtrC and/or other PNNL-provided redox proteins using different electrodes, Fe-containing mineral surfaces, and redox/pH conditions (along with optical absorption characteristics) to establish the conformational state of the adsorbed, electron-transducing protein on Fe(III) oxide surfaces. The electron transfer kinetics of the adsorbed proteins will be spectroscopically determined and compared to calculated electron transfer kinetic parameters (by K. Rosso, a PNNL BGC participant).
3) Orientation and Conformation of Adsorbed Cytochromes as Determined by Second Harmonic Generation (SHG)
Dr. Egglestons' group will perform second harmonic generation (SHG) measurements utilizing i.) a Nd:YAG 10Hz laser coupled to an optical parametric oscillator (at the University of Wyoming) and ii.) a 76 Mhz Ti;sapphire laser (at EMSL). The 76 Mhz measurements will be performed in collaboration with EMSL staff and are not part of this subcontract. Incoming high intensity laser light in SHG is converted to a few photons of doubled-frequency light at noncentrosymmetric interfaces between centrosymmetric materials (e.g., solution and a noncentrosymmetric oxide), providing information about in-situ molecular orientation at the interface. SHG measurements will be performed on adsorbed cytochromes at different pH values to understand how interfacial orientation changes with protein and surface ionization. SHG resonance studies using the Soret band near 400 nm will also be performed to develop additional information on the conformational state of the protein.
Anticipated contract period: November 1, 2004 through September 30, 2007.
Project Details
Project type
Grand Challenge
Start Date
2005-02-22
End Date
2008-10-01
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Lower BH, R Yongsunthon, L Shi, L Wildling, HJ Gruber, NS Wigginton, CL Reardon, GE Pinchuk, T Droubay, JF Boily, and SK Lower. 2009. "Antibody recognition force microscopy shows that outer membrane cytochromes OmcA and MtrC are expressed on the exterior surface of Shewanella oneidensis MR-1." Applied and Environmental Microbiology 75(9):2931-2935.