How E. coli produces hydrogen

原核生物是地球上最简单的生物。它们包括单细胞细菌和它们的表亲古细菌，古细菌是现存的最接近最早生命形式的例子。许多这样的生物可以在没有氧气的情况下生长，而是利用环境中的其他化学物质来产生生命所需的能量。有时使用的化学物质是不寻常的，细菌可以使用宇宙中最简单的分子之一来获得生长所需的能量；氢。此外，一系列微生物，从光合藻类到严格意义上的厌氧细菌，实际上都能产生氢气作为副产品。例如，在缺乏氧气的情况下，一些细菌，如居住在肠道的大肠杆菌，通过发酵的过程生长。这一过程最初会产生甲酸，而甲酸最终被电池用来产生氢气。这个过程需要一种叫做甲酸氢解酶（FHL）的复杂酶的作用，这种酶由至少七种不同的蛋白质以及铁、硫、镍和钼原子组成。早在1932年，英国皇家学会第一位女会员Marjory Stephenson就首次描述了大肠杆菌FHL的活性。在随后的几年里，没有科学家能够分离出FHL，以便更深入地研究它。在这项研究中，我们现在描述了一种创新的新方法，该方法首次纯化了FHL。该项目的总体目标是了解FHL在分子水平上是如何工作的，并修改这种活性，使其适合工业应用。生物制氢方法（所谓的“生物氢”）正变得越来越重要，因为化石燃料资源接近经济开采的极限，碳排放对环境的影响也得到了人们早该认识的认识。氢的单位重量能量是所有燃料中最高的，而且它的使用（特别是在燃料电池中）是清洁和高效的。目前，99%的氢是通过重整化石燃料产生的，1%来自电解，其中大部分被化学工业用作原料。最重要的是，生物氢提供了完全可再生氢的前景，摆脱了对化石燃料的依赖，开发这种资源的空间是巨大的。产氢的生物化学过程依赖于通常被称为氢化酶的对氧敏感的酶。FHL含有一种氢化酶（所谓的“氢-3”酶），它负责大肠杆菌产生的所有氢。氢-3的活性位点含有镍、铁、一氧化碳和氰化物分子（可以使用牛津大学提供的先进光谱学进行研究），因此被称为[NiFe]氢化酶。事实上，我们和其他人已经提出，这种氢化酶的活性位点在氢化学中与铂催化剂一样活跃——铂催化剂是一种昂贵而有限的资源。氢-3在氧气作用下会迅速失活，这可能是长期以来将其分离出来一直存在问题的原因。我们最近对[NiFe]氢化酶的研究，以及对其他酶的研究，已经确定了一个重要的酶子集，可以在空气中起作用（所谓的“耐氧氢化酶”）。这些酶是生物氢技术发展的关键，我们现在对它们的耐氧分子机制有了新的认识。因此，该项目的另一个重要目标是利用这些新知识来设计FHL的氧耐受性。牛津大学和邓迪大学是极具互补性的。邓迪大学在研究大肠杆菌中氢化酶的分子细胞生物学方面具有专长，牛津大学率先采用生物物理方法研究氢化酶，其中最著名的是蛋白质薄膜电化学（PFE）和光谱学。PFE是所有研究氢化酶性质的技术中最强大的技术，在理解其化学的机械细节方面起着重要作用。

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.