ARTICLE pubs.acs.org/JPCB Solution-Based Structural Analysis of the Decaheme Cytochrome, MtrA, by Small-Angle X-ray Scattering and Analytical Ultracentrifugation Mackenzie A. Firer-Sherwood,†,|| Nozomi Ando,‡,§,|| Catherine L. Drennan,‡,§ and Sean J. Elliott*,† †Department of Chemistry, Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215, United States ‡Howard Hughes Medical Institute, §Departments of Biology and Chemistry, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States bS Supporting Information ABSTRACT: The potential exploitation of metal-reducing bacteria as a means for environmental cleanup or alternative fuel is an exciting prospect; however, the cellular processes that would allow for these applications need to be better understood. MtrA is a periplasmic decaheme c-type cytochrome from Shewanella oneidensis involved in the reduction of extracellular iron oxides and therefore is a critical element in Shewanella ability to engage in extracellular charge transfer. As a relatively small 333-residue protein, the heme content is surprisingly high. MtrA is believed to obtain electrons from the inner membrane-bound quinol oxidoreductase, CymA, and shuttle them across the outer membrane to MtrC, another decaheme cytochrome that directly interacts with insoluble metal oxides. How MtrA is able to perform this task is a question of interest. Here through the use of two solution-based techniques, small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC), we present the ﬁrst structural analysis of MtrA. Our results establish that between 0.5 and 4 mg/mL, MtrA exists as a monomeric protein that is shaped like an extended molecular “wire” with a maximum protein dimension (Dmax) of 104 Å and a rod-like aspect ratio of 2.2 to 2.5. This study contributes to a greater understanding of how MtrA fulﬁlls its role in the redox processes that must occur before electrons reach the outside of the cell. ’ INTRODUCTION The Gram negative bacterium Shewanella oneidensis belongs to a class of microorganisms that are capable of using a wide range of electron acceptors in the absence of oxygen, spanning organic compounds (e.g., fumarate)1 to insoluble metal oxides2,3 to soluble inorganic species such as uranyl and chromate ions.4,5 In particular, its ability to couple external metal reduction to the oxidation of organic compounds via dissimilatory metal reduc- tion (DMR) chemistry has made this bacterium a prominent candidate for application in bioremediation as well as microbial fuel cells.3,6 Whereas Shewanella organisms appear to have degenerate pathways of electron transfer that enable growth by anaerobic DMR chemistry, several of the multiheme cyto- chromes of Shewanella encoded by the mtrCABDEF gene cluster have been shown to be responsible for its ability to make direct contact with metal oxides.7 12 However, structural elucidation of any of the Shewanella cytochromes associated with the DMR pathway has proven to be elusive to date, aside from the characterization of OmcA by small-angle X-ray scattering (SAXS).13 In this work, we have examined the size and shape of MtrA from S. oneidensis, a decaheme cytochrome that is proposed to provide an essential molecular “wire” that allows for passage of charge from the periplasm, across the outer membrane, to external electron acceptors. In the current model of the DMR pathway schematized by Figure 1, MtrA is one of the multiple multiheme c-type cytochromes (which have heme cofactors covalently bound to the protein backbone through the cysteines found in the CXXCH heme-binding motif) that simultaneously generate a proton motive force across the cytoplasmic membrane, in part through the action of the tetraheme quinol oxidoreductase (CymA), and conduct charge across the periplasmic space and the outer membrane, ultimately to the cellular exterior by a series of decaheme proteins (MtrA, MtrC, and OmcA). Previously, we have used direct protein electrochemistry to demonstrate that electron transfer between these cytochromes is thermodynami- cally favorable,14 existing as a chain of proteins by which long- range electron transfer occurs. These proteins shuttle electrons from the inside of the cell to the outside, where they directly reduce Fe(III) oxides.10,15,16 CymA is postulated to be anchored to the cytoplasmic membrane by a single transmembrane helix and contain a globular head that inserts into the periplasmic space.17,18 With this orientation, it is able to transfer electrons to many other redox partners such as MtrA. Recently, it has been proposed that MtrA and MtrC interact directly and are as- sembled into a MtrCAB complex, using the porin sheath of MtrB as a scaﬀold.19 Finally, MtrC and OmcA are able to interact directly with and reduce extracellular Fe(III) oxides and have Received: April 18, 2011 Revised: August 12, 2011 Published: August 12, 2011 r 2011 American Chemical Society 11208 dx.doi.org/10.1021/jp203603r | J. Phys. Chem. B 2011, 115, 11208–11214