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

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.

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

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.