21 20 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 D I A M O N D L I G H T S O U R C E A N N U A L R E V I E W 2 0 2 0 / 2 1 Macromolecular Crystallography Group Beamlines I03 and I04 Biological wires for plugging cells into the environment Related publication: Edwards M. J.,White G. F., Butt J. N., Richardson D. J. & Clarke T. A. The Crystal Structure of a Biological Insulated Transmembrane MolecularWire. Cell 181 , 665-673.e10 (2020). DOI: 10.1016/j.cell.2020.03.032 Publication keywords: Extracellular electron transfer; Shewanella; Porin-cytochrome complex; Iron oxidation; Metal reduction; Respiration; Outer membrane protein; Electrogenic bacteria; Geobacter; Decaheme S ome bacteria can survive in the absence of oxygen by using a metal, such as iron, instead. To do this, they require a system for getting electrons from the inside of the cell to the outside, which is usually prevented by the insulatingmembrane surrounding the cell. There was no known system that could do this. Previous research identified three genes in the aquatic bacterium Shewanellaoneidensis that couldmove electrons out of the cell. However, it was unclear how these three genes could cause such an effect. Researchers from the University of East Anglia investigated how the products of these genes could assemble into something allowing electron transport across the outer membrane of the cell. They collected X-ray data onMacromolecular Crystallography (MX) beamlines I03 and I04, using intense, tuneable X-rays to collect data from the small and fragile protein crystals. Using this data, the team were able to identify the positions of the 20 iron atoms inside the protein structure, which allowed the building of the complete structure. For the first time, they were able to see how nature assembles wires out of biological material, and how to build complexes to allow electrons to flow into and out of a living cell. Complexes such as this have a range of potential applications, including biomining usingmicrobes to extract copper and gold from low-grade ores. Many microorganisms use extracellular electron transfer (EET) as a mechanismof generating energy from the environment.This process is common in the soils and sediments of the earth’s subsurface, where substrates in the form of metal oxides and metal ions are used in place of oxygen. Microorganisms use EET to generate energy for growth and survival by moving electrons released from the breakdown of organic molecules into substrates outside the cell. This presents a challenge, as these substrates are often insoluble or toxic and cannot be taken into the cell, so electrons have to be transported through the impermeable barriers that cover the cell 1 . EET bacteria such as Shewanella have developed mechanisms to overcome this problem. Shewanella is a gram-negative bacterium that has an outer membrane that surrounds the cell. Cell membranes are insulative and impermeable, meaning that electrons and water-soluble molecules cannot pass through. To move these electrons across the outer membrane Shewanella assembles protein complexes, known as Mtr complexes, in the outer membrane. These function as biological wires to allow electrons to pass to the cell surface where they can react with a range of materials. For Shewanella these have been shown to include iron and manganese oxides, synthetic electrodes, soluble toxic metals such as uranium and chromium as well as organic soil matter such as humus 2 . The Mtr complex was known to consist of three components: a transmembrane barrel-like structure known as a porin, MtrB, and two proteins that contain ten haem groups, which are organic molecules that tightly bind individualironatoms 3 .Theseproteinsareknownascytochromes,asthetenhaem groups give the protein a distinctive blood-like colour. The two cytochromes of the Mtr complex are known as MtrA and MtrC. MtrA is responsible for collecting electrons inside the cell, while MtrC is exposed on the cell surface and is important for the reduction of the majority of extracellular substrates. The arrangement of the three component proteins of the Mtr complex was not clear until a ‘porin-cytochrome’model was proposed 4 .This model suggested that the two cytochromes on opposite sides of the membrane made contact through the porin, allowing for electrons to move across the membrane using the twenty iron atoms in the two cytochromes. However, the absence of a complete structure of the complex made it hard to understand how it could assemble into a conductive wire and catalyse the reduction of a broad range of substrates. In 2020 we published the first structure of a complete Mtr complex, revealing the molecular details of bacterial extracellular electron transfer 5 . TosolvethestructurebyX-rayproteincrystallography,crystalswereobtained for both the complete Mtr complex and the MtrC subunit. X-ray diffraction data were collected on beamlines I03 and I04 at Diamond Light Source where the structures were solved by single-wavelength anomalous diffraction phasing. 20 iron atoms were located for the Mtr complex corresponding to the 20 haems of the complexwith a single copy in the asymmetric unit. A higher resolution native dataset was subsequently collected for the Mtr complex giving final resolutions of0.27nm(2.7Å)and0.23nm(2.3Å)fortheMtrcomplexandMtrCrespectively. The structure showed for the first time how the complex was constructed and revealed that MtrA formed an elongated protein which spanned the entire width of the outer membrane (Fig. 1), with MtrB wrapping around MtrA to form a membrane spanning porin cytochrome complex. MtrC is bound to the top of this complex, functioning as a highly exposed‘hub’for electron transfer to extracellular substrates (Fig. 1a). This structure revealed that the MtrA-MtrB complex was the central component required for electron transfer across the outer membrane. The ten haemgroupsofMtrAformedalinear,closelypackedchainofironatoms,allowing rapid electron transport across 8 nm.The outer membrane is approximately ~ 4 nm wide, allowing MtrA to protrude from both sides (Fig. 1b). Because MtrA is water soluble, it cannot pass through themembrane and so is held in place in the outer membrane by MtrB. The interior of the MtrB barrel is water soluble while the exterior ismembrane soluble, this allowsMtrB to function as a collar for MtrA and holds it in place in the outer membrane. Long protein chains from the ends of the MtrB barrel wrap around the exterior facing end of MtrA, preventing the accidental leakage of water or other small molecules from the periplasm, while the cell interior facing end is loose, allowing it to swing freely and interact with electron donors inside the cell. This MtrAB porin-cytochrome conduit is commonly found in bacteria that are capable of EET. This includes both ‘metal breathing’ bacteria and ‘metal eating’bacteria, which take in electrons from the environment and use them to fix carbon dioxide. It is suggested that these bacteriamay have been some of the earliest forms of metabolism on the planet. The homology between the MtrA- MtrB porin cytochrome complexes of metal breathing and metal eating bacteria provides insight into how these two-component complexes can function to accept electrons fromsolublemetals and transfer them into periplasmic electron acceptors. The third component of the Mtr complex from the metal breathing system is MtrC, which binds to the top of the MtrAB complex without embedding within the porin.This means that the majority of the surface area remains exposed and capable of electron transfer to different substrates outside the cell. Unlike MtrA, the ten haems of MtrC are arranged in a planar cross-like configuration, allowing for electrons to enter or leave from four possible sites (Fig. 1c).The complex now reveals that electrons enter MtrC via one haem site. At the opposite end of the structure there is an exposed haem that is raised approximately 10 nm (100 Å) above the surface of the cell. The surface of MtrC is also tilted, allowing partial exposure of the other haems.This orientationwould allow for direct reduction of insoluble minerals and metal through the top exposed haem, as well as soluble metals and electron acceptors. This structure of an Mtr complex has finally resolved the mystery of how bacteriacanmoveelectronsacrosstheoutermembraneofthecell,byassembling a porin-cytochrome complex containing a chain of haems that stretches from one side of the membrane to another. Knowing the position and orientation of the MtrC on the cell surface allows for electron transfer to minerals, electrodes and other electrogenic reactions to be better understood and manipulated. Determining this structure has provided a molecular blueprint which will allow us to think about how to directly interface biological cells to electrical systems, as well as drive and control bothmetabolic and biosynthetic reactions, including synthesis of platform chemicals or efficient processing of wastewater. References: 1. White G. F. et al. Mechanisms of Bacterial Extracellular Electron Exchange. Advances in Microbial Physiology (ed. Poole R. K. B.T.-A. in M. P.) 68 , 87–138 (Academic Press, 2016). DOI: 10.1016/bs.ampbs.2016.02.002 2. Beblawy S. et al. Extracellular reduction of solid electron acceptors by Shewanella oneidensis. Mol. Microbiol. 109 , 571–583 (2018). DOI: 10.1111/ mmi.14067 3. Ross D. E. et al. Characterization of protein-protein interactions involved in iron reduction by Shewanella oneidensis MR-1. Appl. Environ. Microbiol. 73 , 5797–5808 (2007). DOI: 10.1128/AEM.00146-07 4. Hartshorne R. S. et al. Characterization of an electron conduit between bacteria and the extracellular environment. Proc. Natl. Acad. Sci. U. S. A. 106 , 22169–22174 (2009). DOI: 10.1073/pnas.0900086106 5. Edwards M. J. et al. The Crystal Structure of a Biological Insulated Transmembrane MolecularWire. Cell 181 , 665-673.e10 (2020). DOI: 10.1016/j.cell.2020.03.032 Funding acknowledgement: This research was supported by the Biotechnology and Biological Sciences Research Council grants BB/K009885/1, BB/L023733/1 and BB/H007288/1. Corresponding authors: Thomas A Clarke, University of East Anglia,
[email protected]; Marcus J Edwards, University of Essex,
[email protected] Figure 1: Structure of a bacterial wire. ( a ) Structure and position of the Mtr complex in the outer membrane of a bacterial cell. The MtrA, MtrB and MtrC domains are coloured red, yellow and cyan respectively, while the haem chain that passes from the cell interior and extends into the environment is coloured blue; ( b ) Cut-through of the MtrAB porin-cytochrome component) revealing how MtrA (red) passes through the interior of the MtrB porin (yellow); ( c ) Crystal structure of MtrC revealing the cross-like configuration of the haem groups. The haems are shown in blue spheres, except for the haems likely involved in electron input (black) and export (light grey).