How do multi-heme cytochromes form transmembrane wires and conduct electrons between the cell and environment?

Lead Research Organisation: University of East Anglia
Department Name: Biological Sciences

Abstract

Humans obtain the energy they need for life by respiring ('breathing') oxygen. This process involves using electrons extracted from the food we eat to convert oxygen to water in a process known as oxygen reduction. Free energy is released in this process and we use this to make ATP, which is the universal energy currency of life. Our dependency on oxygen makes us obligate aerobes -take away the oxygen and we die. Thus humans are confined to living on the surface of planet Earth where oxygen is freely available. However, the vast proportion of Earth's habitable environments are not exploited by humans, but by a diversity of micro-organisms, including bacteria, that can live in the absence of oxygen. What is truly amazing is that some of these bacteria can live deep in the Earth's subsurface and survive by 'breathing rocks'. This is because some of the most abundant respiratory substrates in the Earth's subsurface environments are insoluble minerals, particularly minerals of iron. Such minerals give some soils a reddish colour and they can also be seen as red seams in exposed cliffs. In fact 'iron respiration' is amongst the most widespread respiratory process in anoxic zones and so has wide environmental significance. For example it directly impacts on the balance of several biogeochemical cycles such as the nitrogen, sulphur and carbon cycles and this can in turn influence the release of potent greenhouse gases, such as nitrous oxide. It can also be detrimental to the oil industry through contributing to the dissolution of subsurface or submarine oil pipes. In some aspects the way bacteria respire mineral iron is similar to the way in which they respire oxygen, using electrons to 'reduce' the respiratory substrate. Thus, electrons generated by metabolism inside the bacterial cell are passed to the iron, which changes its electronic state from a so-called 'ferric state' to a 'ferrous state' by the negatively charged electron. However, because the ferric iron mineral is a large insoluble particle it cannot freely diffuse into bacterial cells. Consequently, if a bacterium is to be able to utilise an iron mineral as a respiratory electron acceptor it must have a molecular answer to a perplexing question. 'How can the bacteria move electrons to the outside of the cell where the mineral is located when the electrons are generated by cellular metabolism inside the cell?' This is a very challenging problem for a so-called Gram negative bacteria since they are surrounded by two sealed cell membranes, the inner membrane and the outer membrane, and the insoluble mineral iron lies outside of this outer membrane. Part of the solution to the problem lies in special 'electron transfer proteins' that actually sit on the outside of the cell where they can pass electrons to extracellular insoluble minerals. However, this is not the whole solution, since there still needs to be a specialised electron transfer system to take the electrons across the outer membrane to mediate the passage of electrons out of the cell to these cell-surface proteins. The mechanism by which this electron transfer out of the cell to the so called 'microbe-mineral interface' occurs is still not known. It represents a major question in the study of the biochemistry of an environmentally abundant group of bacteria. Answering it will provide new insights into bacterial energetic processes. It will also have important biotechnological impacts since there is potential for using mineral oxide respiring bacteria in bioremediation processes for the clean up of environments contaminated with toxic organic pollutants (e.g. oil leaks) or radioactive metals, such as Uranium (VI). Their use in microbial fuel cells where the bacteria can be used to generate electric currents using electrodes as solid extracellular electron acceptors is also being explored.

Technical Summary

In the absence of oxygen, some bacterial species can couple oxidation of organic matter to reduction of oxidized metals, such as iron [Fe(III)] and manganese [Mn(IV)] (hydr)oxides, via a biological process termed dissimilatory metal reduction (DMR) that can be coupled to energy conservation. It is becoming increasingly apparent that this process is not confined to the reduction of insoluble minerals, but is also used by some bacteria to reduce or oxidise soluble electron acceptors into insoluble precipitates. The electron transfer pathway of these bacteria requires a number of multi-heme cytochromes that are assembled in the periplasm and transported to the outside of the cell, but key questions are yet to be resolved, in particular how electrons are passed through the outer membrane to the extracellular multi-heme cytochromes and ultimately to the mineral surface. We propose that a system typically consisting of a two-protein core containing an integral membrane 24-28 strand porin and a periplasmic decaheme cytochrome is responsible for electron transfer through the membrane. In addition, a cell surface associated extracellular oxido-reductase can also be present. In this programme we will solve the structures of two decaheme cytochromes from either side of the membrane and these will help to understand the way in which electrons are transferred across the outer membrane. Surface mapping experiments will allow the extent to which the two cytochromes are buried within the porin to be evaluated and confirm whether direct electron transfer between the cytochromes is possible. Finally the topology and ways in which the porin interacts with the intracellular and extracellular cytochromes will be investigated leading to a full structural understanding of this electron transport system. This research would illuminate the first, and therefore paradigm, model for this novel mechanism so important for understanding how bacteria interact with their environment

Planned Impact

This research aims to uncover a novel and ubiquitous porin-cytochrome electron transfer complex (PCET) that functions as an outer membrane 'wire' and allows electrons to move from within the cell to the extracellullar environment. As such it has the potential for involvement in multiple reaction pathways that will influence both bacterial survival and also the environment as a whole. Consequently it is an important pathway that will be of significant interest to a diverse range of scientists and engineers both on the short-tem and long-term timescale. The PCET will be of immediate importance to microbiologists and biochemists as they uncover the properties of bacteria that allow them to survive in anoxic conditions. Biogeochemists will also be able to use this research to identify how bacteria affect the content of minerals such as iron and manganese oxides in the environment . Due to the importance of this system in several transition element cycles, it also influences other inorganic cycles, such as the nitrogen and sulfur cycles. As such it will be of long term importance to environmentalists who seek to understand how bacteria can effect global elemental cycles. Many of the bacteria that employ this PCET system are responsible for the corrosion of metals in sub-soil and aquatic environments. This research therefore is also of immediate importance to engineers and the oil industry as these bacteria contribute to the dissolution of subsurface or submarine oil pipes. This is reflected in the interest of Schlumberger in this research, who have have provided a letter of support. Metal reducing bacteria have also been explored for their potential in bioremediation using Fe(III) as electron acceptors. Shewanella metabolism could have several important biotechnological impacts since there is potential for using mineral oxide respiring bacteria in bioremediation processes for the clean up of environments contaminated with toxic organic pollutants (e.g. oil leaks) or radioactive metals, such as Uranium (VI). In a microbial fuel cell (MFC) bacteria extract electrons from organic substrates and pass them to electrodes such that an electrical current is produced. In many prototype cells the electrons pass via soluble artifical electron carrier mediators that are both costly and often toxic. The prospect then of using bacteria that can pass electron directly to electrode surfaces by virtue of possessing the extracellular electron transport proteins we have characterised in an attractive one. Furthermore it is also a feature of Shewanella that they are metabolically very versatile and can catabolise a wide range of organic substrates and potentially extract electrons from a wide-range of wastes. The goal now is to explore the capacity of different species of shewanella to pass electrons from a range of organic substrates to electrode surfaces. Shewanella is non-pathogenic and the diversity of its metabolism means that there is no need to engineer any new function into it to enable it to transfer electrons from organic carbon to electrode surfaces. The work has international out-reach through our collaborations with researchers in the Pacific Northwest National Laboratory, Richland, USA (see letter of support) that have evolved out of a US Department of Energy Biogeochemical Grand Challenge. Researchers working in the UEA laboratories benefit from these international interactions through regular video conferences and meetings. We also have regular contact with scientists working for Schlumberger and will continue to collaborate through the successful funding of this work. The successful outcomes of these goals will be disseminated through oral and poster presentation at national and international meetings, as well as publication in peer reviewed journals.

Publications

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Clarke TA (2011) Structure of a bacterial cell surface decaheme electron conduit. in Proceedings of the National Academy of Sciences of the United States of America

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Richardson DJ (2013) Controlling electron transfer at the microbe-mineral interface. in Proceedings of the National Academy of Sciences of the United States of America

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Richardson DJ (2012) The 'porin-cytochrome' model for microbe-to-mineral electron transfer. in Molecular microbiology

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White GF (2016) Mechanisms of Bacterial Extracellular Electron Exchange. in Advances in microbial physiology

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White GF (2013) Rapid electron exchange between surface-exposed bacterial cytochromes and Fe(III) minerals. in Proceedings of the National Academy of Sciences of the United States of America

 
Title Video describing work on microbial wires 
Description Video generated by the University of East Anglia describing the work of the group funded by the BBSRC. URL given below; http://www.youtube.com/watch?v=2AXUpi8YFqg 
Type Of Art Film/Video/Animation 
Year Produced 2012 
Impact no actual impacts realised to date 
URL http://www.youtube.com/watch?v=2AXUpi8YFqg
 
Description It was known for over 20 years that bacteria were able to interact with rocks and use them as an alternative to oxygen in a process known as mineral respiration or 'rock breathing', however, it was unclear how the bacteria were able to do this. Over the three years of this project we investigated a potential 'molecular wire' that may be involved in this process. During this project we obtained the structures of two different proteins from the surface of the bacteria and showed that they had ten iron atoms held by haems and arranged in a unique 'staggered cross' motif. These new structures gave insights into how these cytochrome might form the connection between bacterial surface and mineral.

It was proposed that these proteins were able to transfer electricity to the mineral surface by direct contact. In order to prove this we assembled synthetic membrane capsules called 'vesicles' and inserted the molecular wire into their membrane. It was possible to fill the vesicle with electric charge and then measure the rate of discharge through the molecular wire into minerals. Thus we were able to prove that this molecular wire was able to directly interact with the surface of minerals and also suggest ways that it may happen using the new structural data.
Exploitation Route Vesicle model system can be used by industrial companies who wish to develop electrode coatings for adhesion of bacteria to electrodes. Structures can be used develop molecular tethers that can attach the bacteria to electodes and help to understand how nanoparticles can be generated by these proteins. Both the structures and model vesicle systems can be used to look at how to connect bacteria more effectively with electrodes. This will improve the potential for bio-batteries in the future. These systems will also help us understand how to put electrons back into the bacteria, allowing the possibility of generating useful chemicals in the future. The structures have revealed a conserved family of proteins on the surface of these mineral respiring bacteria, that have been shown to be important in interacting with minerals and other external substrates.
Sectors Chemicals,Energy

 
Description As part of an educational resource pack for Biology A-level teachers.
First Year Of Impact 2015
Sector Education
Impact Types Societal

 
Description BBSRC
Amount £476,441 (GBP)
Funding ID BB/P01819X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 01/2018 
End 12/2020
 
Title MtrF 
Description Crystal structure of first outer membrane cytochrome MtrF 
Type Of Material Database/Collection of data 
Year Produced 2011 
Provided To Others? Yes  
Impact Allowed the first molecular interaction between biological and mineral substances to be considered. 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=3PMQ
 
Title OmcA 
Description Crystal structure of outer membrane cytochrome OmcA 
Type Of Material Database/Collection of data 
Year Produced 2014 
Provided To Others? Yes  
Impact allowed other researchers to model interactions and electron transfer through this outer membrane cytochrome 
URL http://www.rcsb.org/pdb/explore/explore.do?structureId=4LMH
 
Description BBSRC CASE partnership with Schlumberger 
Organisation Schlumberger Limited
Department Schlumberger Cambridge Research
Country United Kingdom 
Sector Academic/University 
PI Contribution As part of this work a BBSRC CASE has been awarded with Schlumberger as an Industrial CASE partner. This studentship aims to generate formate from CO2 using a microbial fuel cell
Start Year 2013
 
Description UEA Open day demonstration 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact Approximately 300 prospective undergraduates and parents attended a series of open days where we ran a demonstration of microbial electricity. Many students expressed interest in learning more about how microbes can produce electricity, dissolve minerals, and form wires.
Year(s) Of Engagement Activity 2013,2014,2015
 
Description USAF focussed meeting (University of Southern California) 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Policymakers/politicians
Results and Impact Discussion has led to a working document to facilitate possible research relevant to the US airforce

After the discussion we have now formed links with several US groups who are interested in collaboration.
Year(s) Of Engagement Activity 2014