Prokaryotes are the simplest living organisms on planet Earth. They include the single-celled bacteria and their cousins the archaea, which are the closest surviving examples of the earliest life-forms that ever existed. Many of these organisms can grow without oxygen, and instead utilise other chemicals from the environment to generate energy for life. Sometimes the chemicals used are unusual and bacteria can use one of the simplest molecules in the Universe to gain energy for growth; hydrogen. Moreover, a range of microorganisms, from photosynthetic algae to strictly anaerobic bacteria, can actually produce hydrogen as a by-product. For example, in the absence of oxygen some bacteria, such as the gut-dwelling Escherichia coli, grow by a process known as fermentation. This initially results in formic acid being produced, which is ultimately used by the cell to generate hydrogen gas. This process requires the action of a complicated enzyme called formate hydrogenlyase (FHL), which comprises at least seven different proteins together with iron, sulphur, nickel and molybdenum atoms. The activity of E. coli FHL was first described as long ago as 1932 by Marjory Stephenson, the first female Fellow of the Royal Society. In the years that have followed, no scientist has been able to isolate FHL in order to study it more closely. In this research, we now describe an innovative new approach that has allowed the purification of FHL for the first time. The overall aim of this project is to understand how FHL works at the molecular level, and modify this activity so it will be suitable for industrial applications. Biological approaches to hydrogen production (so-called 'biohydrogen') are growing in importance as fossil fuel resources verge on the limits of economical extraction, and the environmental impact of carbon emissions gains long-overdue recognition. Hydrogen has the highest energy per weight of any fuel, and its use (particularly in a fuel cell) is clean and efficient. At present 99% of hydrogen is produced by reforming fossil fuels and 1% comes from electrolysis, with most being used as a feedstock by the chemical industry. Most importantly, biohydrogen offers the prospect of FULLY RENEWABLE hydrogen, freed from any dependence on fossil fuel, and the scope for taping into this resource is enormous. The biochemistry of hydrogen production depends upon normally oxygen-sensitive enzymes known as hydrogenases. FHL contains a hydrogenase (the so-called 'Hyd-3' enzyme) that is responsible for all of the hydrogen produced by E. coli. The active site of Hyd-3 contains nickel, iron, carbon monoxide and cyanide molecules (which can be studied using the advanced spectroscopy available in Oxford), and is thus termed a [NiFe]hydrogenase. Indeed, we and others have proposed that the active sites of such hydrogenases are as active in hydrogen chemistry as platinum catalysts - an expensive and limited resource. Hyd-3 is rapidly inactivated by oxygen, and this may be a reason why its isolation has proven problematic for so long. Our recent studies of [NiFe]hydrogenases, together with that of others, has identified an important subset of enzymes that can function in air (so-called 'oxygen-tolerant hydrogenases'). These enzymes hold the key to technological developments of biohydrogen and we now have fresh insight into the molecular mechanism of their oxygen tolerance. Another important aim of this project, therefore, is to use this new knowledge to engineer oxygen tolerance into FHL. The Oxford and Dundee groups are superbly complementary. Dundee has expertise in studying the molecular cell biology of hydrogenases in E. coli, and Oxford has pioneered biophysical methods for studying hydrogenases, most notably protein film electrochemistry (PFE) and spectroscopy. PFE is the most powerful of all techniques for studying the properties of hydrogenases and has been instrumental in understanding the mechanistic details of their chemistry.

Technical Abstract:
Escherichia coli is a bacterium with a flexible metabolism and renowned genetic tractability that has established it as an important 'model', or 'chassis', organism for biotechnologists interested in genetically modifying metabolism, or even designing completely synthetic activities. E. coli can perform a 'mixed-acid fermentation' in which glucose is metabolised to ethanol and various organic acids, including formate. This formate is further disproportionated to carbon dioxide and hydrogen by the formate hydrogenlyase (FHL) complex. The scope for tapping into this activity is enormous since it offers the possibility of fully renewable biohydrogen, freed from any dependence on fossil fuel. Although the genetics and physiology of FHL are understood, the instability and oxygen-lability of this important enzyme have meant that it has never been isolated in an intact or active form. In this proposal, we describe an innovative genetic solution to this problem and demonstrate the isolation of FHL for the first time. Our isolation of FHL is a major breakthrough in the fields of bioenergetics, membrane biology, and bioenergy research. An intensive, fully complementary, and cost-effective program of studies is thus planned to address the molecular basis of biohydrogen production by E. coli. FHL is a membrane-bound multi-enzyme complex comprising a formate dehydrogenase and a [NiFe]hydrogenase. The Dundee/Oxford collaboration offers superbly complementary approaches to the characterisation of FHL. The Dundee group has internationally-recognised expertise in cell and molecular biology of E. coli hydrogenases, and in the biochemistry of bacterial membrane proteins. The Oxford group have pioneered protein film electrochemistry for studying hydrogenases, which has now identified the molecular basis of the oxygen-tolerance mechanism utilised by some [NiFe]hydrogenases, and are world-leaders in unravelling the mechanisms of complex metalloenzymes using advanced spectroscopy.

Potential Impact:
In recent years, biological approaches to energy production (so-called 'bioenergy') are growing in importance as fossil fuel resources verge on the limits of economical extraction, and the environmental impact of carbon emissions gains long-overdue recognition. Hydrogen gas is among the most exciting of the current options for future energy needs. As the immediate product of energizing water by photolysis (sunlight) or renewable-powered electrolysis, or via biological routes (photosynthetic algae or 'dark' fermentation of waste materials), hydrogen is potentially the 'greenest' and most renewable of fuels. Currently, hydrogen is predominantly used as a feedstock in the chemical industry - but it is increasingly being used as a fuel. Hydrogen as a fuel is being championed by advanced countries, particularly through the governments of the USA, Australia, Germany and Sweden. Although the drawbacks of hydrogen are frequently aired (e.g. low energy density, storage difficulties, primitive supply and distribution infrastructure) these issues cannot be allowed to hold back its research and development, particularly in an academic context. 'Needs must' and hydrogen (including biohydrogen) will eventually become a dominant part of human lives and economies. Presently, 99% of hydrogen is produced by reforming fossil fuels. This approach is unsustainable and alternative, fully renewable, solutions must be found. This research project focuses on the mechanism of hydrogen production by Escherichia coli - the model organism of choice for biotechnologists the world over. Multinational energy companies, as well as innovative SMEs, will be greatly interested in the breakthroughs offered by this research. Industrialists who specialise in bioprocessing will also benefit from this work since we will be exploring methodology for the isolation of multi-enzyme, and membrane-bound, protein complexes. Biomedical companies will also be interested in this study of bacterial hydrogen metabolism, since hydrogenases are key virulence factors in pathogens related to E. coli - most notably Salmonella. Researchers interested in the emerging field of 'synthetic biology' should also be interested in this work. The principles of synthetic biology, some of which are applied here, can help overcome many hurdles in basic biological science research. This is the way forward for molecular biology. The staff trained on this project will be of immediate great interest to the industrial biotechnology and biomedical sectors. The project constantly applies electrochemistry to addressing biological questions, and this is a key modern technique at the interfaces of chemistry, physics and biology. Experts in this area will be in high demand, especially for companies interested in developing biosensors and other hybrid devices. Workers skilled in modern molecular biology, as well as membrane protein biochemistry, will also be in immediate demand. The Universities of Dundee and Oxford work hard to provide generic skills training in a broad spectrum of areas so that scientists are ready and able to contribute to the UK economy in whatever future career they so choose. Scientists at both institutions are exposed to a wide range of subjects, and the latest cutting-edge technology, so cannot fail to acquire both a strong work-ethic and broad understanding of the sciences. Overall, this project will benefit the nation's wealth as we move to a low carbon economy over the next 10-20 years. It will improve the nation's health over the same timescale as the use of hydrogen as a fuel increases (zero toxic or environmentally harmful emissions). It at will also place UK science at the cutting edge as the leading player in global biohydrogen research and development.