Sparks of Life

Chemists are well acquainted with redox reactions, as many processes involve the transfer of electrons from one atom or molecule to another. Redox reactions are of great interest to biologists as well, since most of the network of reactions that make up the metabolism of a cell also depend on electron transfer. One particular assembly of redox reactants, the electron transport (ET) chain, generates energy for the cell. Forty years after their discovery ET chains are once again at the frontier of biochemical research. Two particularly fascinating fields are the construction of biofuel cells and the design of artificial ET chains.

Redox for life

When electrons are transferred between atoms or molecules the acceptor is reduced, whilst the electron donor is oxidised, so that the process as a whole is called a redox reaction. Combustion is one common example. When glucose combusts completely the carbon atoms are oxidised to carbon dioxide gas and the hydrogen to water. The oxygen atoms in O₂ accept electrons and so are reduced:

C6H12O6 + 6O2 ------> 6CO2 + 6H2O + energy

(reductant)    (oxidant)

Redox reactions are especially important in biochemistry. The metabolic pathways of a cell are the series of chemical reactions that it needs to survive, and most of these involve the transfer of electrons. One set of reactions, called respiration, occurs continuously in all living cells to provide them with usable energy. Bacteria perform these reactions themselves, but animal or plant cells rely on organelles within them called mitochondria to undertake respiration.

During respiration, complex carbohydrates such as glucose are carefully broken down one bond at a time. Each time the fuel molecule is oxidised, by the removal of a carbon or hydrogen atom, or the addition of oxygen, the released electrons are collected by a protein called an electron carrier. The electron carriers take their load to another protein, which is embedded within the membrane of the bacteria (or mitochondrion). The electrons have been delivered to the first component of a whole sequence of oxidising agents known as the electron transport (ET) chain. The electrons are transferred from protein to protein down the ET chain, each protein oxidising the one before it. A few of the proteins along the way use some of the electron's energy to pump an H+ ion (a proton) across the membrane and out of the cell. These protons are allowed to flow back across the membrane through a protein that synthesises ATP, the cell's energy storage molecule.

At the end of the transport chain the electrons are passed to the final electron acceptor. Some cells reduce O₂ to water, which is known as aerobic respiration. But in anaerobic conditions, when oxygen is not available, another acceptor must be found. Figure 1 illustrates the process of aerobic respiration.

     
Figure 1. Glucose metabolism and the ET transport chain of bacteria and mitochondria

So instead of heat being produced in respiration, as it would if the glucose had simply been burned, the cell has controlled the reaction and stored useful energy in the form of ATP:

 

C6H12O6 + 6O2 -----> 6CO2 + 6H2O + 30ATP 

(reductant)                      (oxidant)                 (energy) 

 

The chemistry of redox reactions has therefore been developed into extremely sophisticated electrochemical systems by evolution. Forty years after their discovery these electron transport chains are again at the very cutting edge of biochemical research. We will look at two examples here: building microbial fuel cells and designing artificial transport chains for nanotechnology.

Microbial Fuel Cells

Fuel cells rely on the redox reaction between replenishable agents to generate electricity. The reducing agent passes its electrons to the anode, they produce a current in the circuit, and then reduce the oxidant at the cathode. For example, NASA's space shuttle runs on hydrogenoxygen fuel cells, but in principle any redox pair could be used.

As we have seen, bacteria make very efficient use of redox reactions in their respiratory pathways and ET chain. Would it be feasible to build a biofuel cell of bacteria in a carbohydrate solution? Such microbial fuel cells were first proposed in the mid-1980s, but scientists were never able to achieve a reasonable efficiency. It is only very recently, however, with the discovery of a new species of bacteria and the publication of the latest experiments last month, that this finally seems practical.

It began with a routine scientific expedition to search for previously unknown bacterial species, dredging for samples in the bottom sediment of Oyster Bay in Virginia, USA. The silt samples were taken to a microbiology laboratory and analysed to see what kinds of microbes were growing in them. Several novel bacteria species were discovered and then investigated more closely in order to characterise them. Tests were done to reveal details of their metabolism, such as which nutrient molecules they live off and what the final electron acceptor in their respiratory pathway is. As a result of this, one of the new bacteria was classified as a ?facultative anaerobe that uses Fe³⁺ ions as an electron acceptor?. This means that although the bacterium can respire with oxygen, it is also able to survive in anaerobic conditions. Oxygen is often rare in marine sediments because it is used up by respiring organisms and diffuses back in very slowly. Oxidised metallic ions are relatively abundant in sediment, however, especially near river estuaries where metals from rocks have dissolved into the water and reacted with oxygen.

This newly discovered bacterium has therefore evolved the ability to use oxidised iron instead of O₂ at the end of its electron transport chain and Fe³⁺ ions are reduced to Fe²⁺. The bacterium has now been named Rhodoferax ferrireducens in light of this method of respiration. Although other iron-reducing bacteria are known, R. ferrireducens has other properties that may help it to revolutionise organic fuel cells.

Biofuel cells generally consist of two compartments separated by a porous membrane. Bacteria are added to the anode side, which usually colonise the surface of the electrode itself rather than float in the liquid medium. The bacteria respire and oxidise the carbohydrates provided in this medium. The released electrons are passed to the anode, and then onto an oxidising agent (usually oxygen) in the cathode compartment.

There are, however, three main problems with present biofuel cells: mediators, incomplete oxidation, and lack of long-term stability. The mediator is an additional electron carrier that must be added to most biofuel cells. Mediator molecules take electrons directly from the transport chain before they reach the final electron acceptor. They ferry the electrons to the anode where they are oxidised and are then available to collect more. But this extra step makes the fuel cell very inefficient, and typically less that half of the electrons produced by the oxidation of the carbohydrate are transferred to the anode. R. ferrireducens, however, can use Fe(III) as the final electron acceptor, which is itself ideal for shuttling electrons to the anode. This capability has been exploited by Derek Lovley and his team at the University of Massachusetts, USA, in a R. ferrireducens biofuel cell, as shown in Figure 2. The results from this experiment were published in the journal Nature Biotechnology last month. 

 
Figure 2. A biofuel cell using R. ferrireducens.

Furthermore, many conventional biofuel cells do not completely oxidise the carbohydrate so much of the potentially available energy is lost. For example, complete oxidation of glucose yields 24 electrons, but some biofuel cells can only convert glucose to gluconic acid, and so release just two of these electrons. Living fuel cells can also be unstable, and often shutdown when their population of bacteria dies out. R. ferrireducens, on the other hand, is astoundingly efficient and releases as many as 20 of the available electrons. The bacteria use the remaining electrons to create ATP as described above, which means they can grow and multiply even whilst generating electricity. The resulting biofuel cell is very stable and also easy to ?recharge?. Once most of the glucose has been used the whole mix is merely flushed out and replaced with fresh solution. Surviving bacteria quickly multiply to replenish the population and bring the cell back up to maximum output.

Tests performed in small glass beakers with electrodes the size of playing cards have produced 3.5x10^-4 Watts of electrical power. This is still a nascent technology however, and the generating capability of such biofuel cells could be multiplied many-fold. Simply increasing the size of the biofuel cell would scale-up the current produced, and flat electrodes could be replaced with thick blocks of graphite foam. These have an enormous surface area for bacteria to settle on and pass electrons immediately to the anode.

R. ferrireducens has one more advantage in that it is able to metabolise a wide range of simple carbohydrates.

"Using biofuel cells to generate electricity from organic waste matter is finally seeming feasible"

It doesn't need refined or pre-processed nutrient broth, and so general organic waste from farms, homes or industry can be handled by this type of biofuel cell. Instead of being incinerated or dumped into landfills, our waste could be used to generate electricity and help alleviate the problem of carbon emissions and global warming. Since carbon dioxide would be released anyway by the burning or rotting of this waste, we may as well use it to generate electricity and reduce demand on power stations. Dr Loveley is certainly optimistic, and believes that "using biofuel cells to generate electricity from organic waste matter is finally seeming feasible".

Biofuel cells are therefore an extremely effective use of the bacterial electron transport system as a whole. Other biochemists, however, are working with the individual proteins that make up an ET chain.

Engineered electron transport systems

All redox couples have a standard potential - the value representing the strength of an oxidising or reducing agent that is used to calculate the voltage produced by an electrochemical cell. Redox potentials can even be determined for proteins in the ET chain. For example, ubiquinone has a potential of +0.10 V, whereas the value for cytochrome c, which is further ?downstream? in the ET chain, is +0.22 V. As expected, cytochrome c is the stronger oxidising agent, since each protein in the chain must be able to take the electrons from the one before it.

The standard potential is governed by the chemical bonds between amino acids, the units that make up proteins. By changing a few of the amino acids it is possible to modify how strong an oxidant the protein is. This is exactly what a group of biochemists at Imperial College in London, UK, are now doing. Using genetic engineering, Gianfranco Gilardi and his research team randomly mutate the gene for a particular ET protein, so that several amino acids are altered. They then select from the proteins produced those that have a particular standard potential. The result of this is that they can effectively tune the oxidising strength of an ET chain protein to any desired value.

Another ground-breaking technique that Dr Gilardi and his team are developing has been dubbed ?molecular Lego?. Many complex proteins are made up of different modules or domains, each of which performs a different function. The aim of this research is to be able to select domains from separate proteins, and fuse them together into a new ?chimera? protein with the required properties. For example, Dr Gilardi's lab has been working with a protein called cytochrome P450. P450 has three separate domains, including one that contains an iron atom and catalyses certain oxidation reactions. This haem domain has recently been joined with the electron transfer domain from another protein, called flavodoxin. If these synthetic proteins are fixed to the surface of an electrode, a tiny current flows all the while that the haem domain is catalysing the oxidation of its target molecules. This would not be exploited to generate electricity as in the biofuel cell above, but instead can be used to detect whether the catalytic haem domain is currently active or not. Enzymes are very specific catalysts, and only operate on particular target molecules. Therefore a current is only generated if the target compound is present around the electrode, and this effect can be used to build extremely sensitive biosensors. To make such a sensor, you first need to select an enzyme that functions on the molecule you are interested in, and then bolt on an electron transport domain using genetic engineering. As described above, the redox potential of the ET module can even be tuned to the exact value needed.

The network of redox reactions created by evolution has recently inspired a great deal of biochemical research. Bacterial electron transport chains are being exploited to generate electricity from waste, and individual redox proteins are being engineered for artificial chains. Molecular Lego is state-of-the-art nanotechnology, and it promises sensitive biosensors that could be built to detect everything from glucose in the blood of a diabetic to bioterrorism agents in the air. Bacteria are commonly only associated with disease and decay, but they may hold the key to facing some of our greatest challenges in the future.

 

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References:

• Chaudhuri, Lovley (2003), Electricity generation by direct oxidation of glucose in mediatorless microbial fuel cells, Nature Biotechnology 21, 1229-1232

• Gilardi et al (2002), Tuning the Reduction Potential of Engineered cytochrome c-533, Biochemistry 41, 8718-8724
• Gilardi et al (2002), Molecular Lego: design of molecular assemblies of P450 enzymes for nanobiotechnology, Biosensors & Bioelectronics 17, 133-145
• Gilardi et al (2001), Manipulating redox systems: application to nanotechnology, TRENDS in Biotechnology 19, 468-476
• Gilardi et al (1998), Engineering multi-domain redox proteins containing flavodoxin as bio-transformer: preparatory studies by rational design, Biosensors & Bioelectronics 13, 675-685