Nature's masterclass

C&I Issue 8, 2015

Evolution invented catalysis so early that it is safe to assume that there would be no recognisable life on Earth without it, only geology. In today’s living organisms, no chemical reaction is allowed to just happen. For every step of every metabolic pathway, there is an enzyme that looks after it. Some of nature’s catalysis needs are filled by RNA enzymes (ribozymes), possibly a vestige of an earlier version of life in which RNA was the only biopolymer. Most are proteins, however, and their catalyst centres consist of their amino acid residues alone, or they can additionally include metal ions or clusters.

In industry, catalysts are used extensively but they rarely resemble those found in nature. Consider, for example, nitrogen fixation. Nature’s catalyst is nitrogenase, an enzyme with metal clusters in its active centre, which always contain iron, and in some species also molybdenum or vanadium, and runs at ambient temperature and pressure. The industrial Haber Bosch process, by contrast, uses high temperature and high pressure – 200bar, 500°C – and an iron catalyst, but has a very poor yield.

Other widely used catalysts, like the converters in cars, rely heavily on expensive noble metals like platinum, palladium and rhodium. All of this suggests that chemistry could learn a thing or two from nature, which after all has more than 3bn years of experience of catalysis.1

Complex chemistry

Harvard chemist Theodore Betley, who was awarded the Kavli Foundation Emerging Leader in Chemistry Lecture at the American Chemical Society’s 2015 meeting in Denver, systematically develops novel catalytic approaches by incorporating aspects gleaned from natural enzymes.

In one successful project, Betley’s group used oxygenases of the cytochrome P450 family. These enzymes contain an iron atom that forms an unstable bond with the oxygen molecule, making the whole construct highly reactive. All cytochrome P450 enzymes work by reducing one of the atoms in O2 to water. With the other oxygen atom, different enzymes of the family have evolved to perform a wide range of different tasks, which is why many species, including us humans, have dozens or even hundreds of such enzymes for specific oxidation reactions.

Betley’s group built a molecular analogue of the cytochrome P450 active site, which makes use of the same electronic mechanism as the oxygen splitting of the natural enzyme. They replaced the O=O molecule with a hydrocarbon chain terminating in an azide group (N3) and bound this to the iron centre; in doing this, they were able to exploit the strong activation effect not only to induce one nitrogen atom to react with one of the otherwise unreactive carbon–hydrogen bonds but also to close the hydrocarbon to form a five-membered cycle, a pyrrolidine.2

An initial problem with the process was product inhibition, ie the product remained bound to the catalyst and thus blocked it from carrying out the same reaction multiple times. Using amine protection agents, however, the researchers were able to release the product from the iron centre and thus achieve true catalysis with multiple turnovers. With this approach, the group produced a wide range of different heterocycles in simple reactions, with nitrogen gas as the only side product.

One major advantage of Betley’s catalysts is that they can achieve reactions involving several electrons at a time. For example, his group demonstrated that a trinuclear iron complex can catalyse reductions of nitrogen compounds, such as hydrazine, requiring two or four electrons.3 The ultimate goal would be to find a simple molecular catalyst that mimics the nitrogenase reaction, which requires six electrons for each nitrogen molecule to be split.

Another promising target for such catalysts is the reduction of carbon dioxide to methane, which also requires multiple electrons. With the right kind of catalyst, artificial photosynthesis could use solar energy to remove excess carbon dioxide from the air and convert it into fuel, redressing the carbon cycle that our activities in the past two centuries have thrown off balance. ‘Our goal is to answer the questions: How do you take the sea of gases that makes up our atmosphere and convert that into liquid fuels?’ Betley said in a press statement. ‘Or how do you remediate greenhouse gases and turn them into something usable for the chemical industry?’

Nature’s fuel cells

One emerging natural catalyst that could become very useful for renewable energy use is the oxygen-tolerant hydrogenase. Hydrogenases are used in nature either to produce hydrogen – as a sink for accumulating reduction equivalents – or to burn it as a fuel. There are two unrelated classes of hydrogenase enzymes with different metal cofactors: [Fe–Fe] hydrogenases are usually involved in hydrogen production; and [Ni–Fe] hydrogenases, which play a part in controlled oxidation reactions. 

Most of the hydrogen processing in either direction occurs in the absence of oxygen. ‘Knallgas’ bacteria, however, which are scattered among the major groups of proteobacteria, aquificae, actinobacteria and firmicutes, can handle oxygen and hydrogen simultaneously, which makes them very interesting as potential hydrogen producers for renewable energies. 

Based on several crystal structures and mechanistic experiments, researchers are now beginning to develop an understanding of how this natural fuel cell works and how it could be harnessed in technology. To-date, the work has focused on the crystal structures of oxygen-tolerant hydrogenases from three species: the soil bacterium Ralstonia eutropha,4 the marine species Hydrogenovibrio marinus,5 and our own gut microbe, Escherichia coli.6

Researchers at UniCat (Unifying Concepts in Catalysis) in Berlin, Germany, have scrutinised the membrane-bound hydrogenase (MBH) from R. eutropha. The constitutive parts required for hydrogenase activity include a large subunit, containing the nickel–iron complex, and a smaller subunit with three iron–sulfur clusters. In the living cell, this small subunit is anchored to a b-type cytochrome embedded in the membrane, and the whole arrangement of three protein molecules comes in triplicate, with the three trimers held together at the membrane-bound bottom end like a bunch of flowers in a vase. This setup shows the best and most oxygen-tolerant hydrogenase activity.7

The researchers have compared the structures of the oxygen-tolerant hydrogenases to the oxygen-sensitive ones but found that there appears to be no simple explanation for their remarkable capabilities. The active site around the nickel–iron complex looks exactly the same in oxygen-tolerant and in sensitive hydrogenases, so they had to look elsewhere.

The three iron–sulfur complexes of the small subunit are arranged in a line and pass on electrons from the active site in the large subunit to the cytochrome in the membrane. The two complexes on the outer flanks of the subunit normally have a cube structure built of alternating iron and sulfur atoms. Thus each iron is bonded to three sulfur neighbours within the cube, and four cysteine residues of the protein offer additional bonding with their thiol groups.

Johannes Fritsch and colleagues at Berlin’s Humboldt University discovered a subtle change in the iron–sulfur cluster closest to the catalytic subunit in oxygen-tolerant MBH. This cluster is an incomplete cube with only three sulfur atoms, which also means that it is much more flexible than the standard version. Two additional cysteine residues saturate the coordination sphere of the iron atoms. It turned out that this is an adaptation to the presence of oxygen. This open, flexible conformation can carry out two redox reactions within the range of physiological conditions and thus provide two electrons in rapid succession, allowing the reduction of molecular oxygen to hydroxide. This reaction helps to avoid the presence of reactive oxygen species, which could damage the molecular fabric of the enzyme.8

The groups of Oliver Lenz at the Technical University and Patrick Scheerer at the Charité Hospital, both in Berlin, reported more detailed investigations into this unusual, open iron–sulfur cluster.9 They solved the crystal structures of MBH in three different redox states and, using spectroscopic and theoretical methods, they found that in the course of its oxidation reaction, the cluster dramatically rearranges its structure. In the most oxidised form, it is bound to a hydroxide ligand that had not been observed before. It remains to be established if the hydroxide indicates that this cluster is directly involved in splitting the oxygen molecule. Alternatively, it may just be the switch that decides in which direction the electrons flow between the active site and the cytochrome in the membrane.   

Meanwhile, the groups of Peter Hildebrandt and Ingo Zebger at the Technical University, have used Raman spectroscopy to characterise the hydrogen splitting reaction at the nickel–iron cluster in the large subunit.10 The variability of the iron–sulfur clusters in MBH is a confounding factor, so the researchers used a simpler hydrogenase from the same organism instead, namely the regulatory hydrogenase (RH). This enzyme also splits hydrogen, but only in order to sense its concentration, and to upregulate the production of the main hydrogenase enzyme when it is needed. Analysing the somewhat simpler electrochemistry of the RH, the researchers could, for the first time, characterise the role of the nickel atom as well as the iron.

All of this information could prove useful for applications in fuel cells. Almost 10 years ago, the groups of Bärbel Friedrich at the Humboldt University in Berlin and Fraser Armstrong at the University of Oxford, UK, succeeded in producing electricity with a fuel cell that uses the hydrogenase from Ralstonia metallidurans.11 Further work in Armstrong’s lab then focused on the hydrogenase Hyd-1 from E. coli as an anode, combined with the enzyme bilirubin oxidase as the cathode, yielding the first artificial fuel cell built purely from biological components.12

An even more promising prospect from a renewable energy perspective would be to couple the oxygen-tolerant hydrogenase with photosystem I to create a biomolecular factory for solar hydrogen.13 Alternatively, chemists like Betley could use the emerging insights into these natural catalysts to create analogous marvels with pure chemistry. 


1 M. Gross, Travels to the nanoworld. New York: Plenum, 1999.
2 E. T. Hennessy and T. A. Betley, Science, 2013, 340, 591.  
3 T. M. Powers and T. A. Betley, J. Am. Chem. Soc., 2013, 135, 12289.
4 J. Fritsch et al, Nature, 2011, 479, 249.
5 Y. Shomura et al, Nature, 2011, 479, 253.
6 A. Volbeda et al, Proc. Natl. Acad. Sci. U.S.A., 2012, 109, 5305.
7 V. Radu et al, J. Am. Chem. Soc., 2014, 136, 8512.
8 J. Fritsch et al, Nat. Rev. Microbiol., 2013, 11, 106.
9 S. Frielingsdorf et al, Nat. Chem. Biol., 2014, 10, 378.
10 M. Horch et al, J. Am. Chem. Soc., 2014, 136, 9870.
11 K. A. Vincent et al, Chem. Commun., 2006, 48, 5033.
12 A. F. Wait et al, J. Phys. Chem. C, 2010, 114, 12003.
13 M. Rögner (ed), Biohydrogen. Berlin: de Gruyter, 2015.

Michael Gross is a science writer based in Oxford, UK

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