Thursday, 7 May 2015

Harnessing light in Biology

A world without colour would be a very different place. Ever since Isaac Newton provided an explanation for the composition of visible light, Scientists from all disciplines have been drawn to colour. Physicists have sought to rationalise the duality of light:particles and waves; chemists have tried to isolate and synthesise coloured molecules for applications ranging from textile dyes to molecular reporters of enzyme function. And Biologists continue to investigate the place of pigmentation in Natural Selection. Personally, I am convinced that colour should be at the core of any Science curriculum. (I wont discuss fluorescence, X-rays, microwaves here, but I may stray into the ultra violet end of the spectrum). So let's consider some of the basics, although our ability to predictably synthesise a molecule from first principles (ab initio) of a specific colour, remains a significant challenge.

As I mentioned above, whether you have a passing interest in Science, or you are a student; or indeed a seasoned campaigner, you should familiarise yourself with Sir Isaac Newton (there are many Biographies to choose from: I like the Last Sorcerer by Michael White). In addition to his work (RHS) on the properties of light (concisely entitled Opticks:or, a treatise of the reflexions, refractions, inflexions and colours of light) Newton made many key contributions to Mathematics and Science. The most relevant point to make here is that visible light comprises a spectrum of colours that can be separated by use of an optical prism. If you are in school, ask your Physics teacher at the next available opportunity, if you can borrow a prism during your next break!. If you haven't carried already out this experiment, no self respecting student of Science (young or old) should delay in acquiring a prism: they cost less than a tenner, if you are in the UK!. This is a classic experiment (in my top ten, which includes Archimedes water displacement and Meselsohn and Stahl's demonstration of the semi-conservative nature of DNA replication) and illustrates that light can be considered as a set of components, in fact the "spectrum" of visible light has wavelengths of between 380-750nm with Violet at the lower wavelength and Red at the upper. The energy associated with the spectral colours is inversely related to the wavelength: the shorter the wavelength the higher the energy. It took around 200 years after Isaac Newton's ideas were published (and of course Newton constructed his ideas, by standing on the shoulders of (other) giants (of Science)), Physicists were beginning to construct atomic models, and the technological advances made in electromagnetism, combined with the ability to produce reliable vacuums, paved the way for the discovery of electrons and electromagnetic radiation. These experiments have provided Chemists and Biologists with a framework for exploring the relationship between light and energy generation in photosynthesis and has been incredibly influential in choosing which molecules to investigate in the field of Biochemistry. As I said earlier, the integration of colour into the "fabric" of life has driven the development of Evolutionary Biology.

Let us consider why copper sulphate is blue in solution, or why blood is red and plants are green. At the first level, drawing on Newton's work, the colours blue, red and green arise because the other colours have been extracted by the solution in the bottle, the blood in our veins and the leaves of the plant. Therefore, since we know that light of different wavelengths possess different levels of energy, it seems straight forward to assume that some of the energy contained within a "ray" of visible light has been removed, or absorbed. This phenomenon of absorption is very well understood by Physicists and usually involves the electrons. Electrons associated with a molecule can, under some circumstances, become excited by a very specific (we say discreet) amount of energy. The electron or electrons are, as a consequence re-organized or redistributed, in the confines of the particular molecule. Hence, the light emerging (say from a solution of copper sulphate) is coloured: some of the energy required to produce visible light has been depleted (or absorbed). In the case of copper sulphate, a Transition metal ion salt, the electrons migrate between the degenerate orbitals extending from the elemental nucleus. In the case of blood and green plants, it is the porphyrin molecule at the centre of haemoglobin, in the  case of blood; or in a modified form that you all know as chlorophyll in plants. In short, electrons in certain molecules are amenable to excitation by visible light and this gives rise to colour. So let's look at some examples of coloured molecules in Nature.

As a first year PhD student in Paul Engel's group at Sheffield in the early 1980s, my senior colleague, Gary Williamson (now Professor of Functional Foods at the University of Leeds) would spend a week every few months growing a foul smelling anaerobic bacterium in our one and only 40 litre glass flask (think indoor glass gardens). The organism shown left, had been described by the first Professor of Microbiology at Sheffield, Sydney Elsden, who later became the Director of the Food Research Institute in Norwich, where incidentally, Gary spent a number of years. It therefore became known as Megasphaera elsdenii. (Those of you with a classical education may wish to contemplate the Professor's sense of humour!). After an overnight bloom, the 40 litres of medium generated a few hundred grams of cell paste, which was then dried and extracted, to produce a protein rich supernatant after centrifugation. The extract was then purified on a white ion exchange column, and a series of brightly coloured protein bands eluted: reds, yellows and for Gary the bright green of the Butyryl-CoA Dehydrogenase. As a relatively "green" Biochemist, I was even greener with envy at Gary's colourful protein: my enzyme: glutamate dehydrogenase was colourless. So why is this important? The colour of a protein is generally a result of a set of interactions between a ligand (a cofactor) and the side chains of the amino acids in the binding site for that ligand. This fortuitous situation provides the Biochemist with an entree into studying the molecular mechanism of action of the protein (in the case of Haemoglobin) or the enzyme in the case of Butyryl-CoA Dehydrogenase. (In fact Gary managed to work out why the enzyme was green in a lovely paper he published while in Paul Engel's lab). Since we know that colour is associated with electron re-organization and the mechanism of action of many enzymes does too, the changes in colour can often "report" on events at the enzyme's active site. This phenomenon is at the heart of much of the Biochemistry published in the last century, from studies on Haemoglobins, Flavoproteins (usually yellow), chlorophyll associated enzymes (often green) and many more besides. I should add that sometimes, it is necessary to use ultra violet light to illuminate enzyme activity, especially in the case of NAD(P)-dependent enzymes, where light is absorbed at a shorter wavelength than violet (typically 340nm, which has a higher energy).

http://www.carolguze.com/images/energy/chlorophyll.jpgTwo other favourite areas for Biochemists are photosynthesis and energy generation. Both involve electron transfer reactions and both are often green or red!. Interestingly the porphyrin ring is a dominant feature of both areas of investigation. Electron rich cofactors provide enzymes with the capacity to catalyse difficult reactions, but the porphyrin ring when complexed by iron or copper has proven to be an evolutionary favourite for facilitating electron transfer along the inner mitochondrial membrane via cytochromes. When the porphyrin ring is complexed by magnesium (see RHS), it is a rich green colour, and, as you can't have failed to notice, is responsible for much of the colour of our landscape. As my colleague at Sheffield, Neil Hunter often tells me, it is one of the most abundant molecules on the planet (certainly the most abundant pigment) and in view of its central role in Life, deserves a little more attention from the research funding agencies. Chlorophyll, along with several other pigments, collaborate with light harvesting proteins and a range of electron transfer molecules to capture, harness and convert light energy (photons) into chemical energy (ATP). Along the way, carbon dioxide is "fixed"in the form of sugars and the plants release the oxygen that we rely upon to complete our own electron transfer process, from which we generate our own ATP. Are the colours red and green of any significance? Not really, they just reflect the levels of energy that are required to excite electrons. Only when there is a matching of excitation energies within the incident light, will absorption events be observed. The observation that discreet amounts of energy are required to promote electrons from one level to another is of course at the heart of quantum mechanics, and the colours of these electron transfer proteins is simply a consequence of this absorption phenomenon. 

Finally moving on to Biology: one of my heroes (and I am not alone!), David Attenborough, has a well known passion for Birds of Paradise; bringing pioneering film of these outrageously colourful birds to our TV screens. Similarly, butterflies and flowering plants are responsible for some of the most striking uses of colour to frighten, attract or hide, and thereby survive; thus presenting naturalists with an opportunity to investigate the selective advantage of pigmentation. Perhaps one of the most interesting case of colour change is found in the chameleon, which is able to apparently change colour in seconds. Unlike the squid, which does a pigment swap, the chameleon does it all with mirrors, or at least with cells capable of altering the reflective properties of their skin. Unlike the biochemical properties of haem proteins and chlorophyll, the colours of insects and flowers arise from biosynthetic pathways whose primary function is often unclear: some of these pathways we refer to as secondary metabolism, mainly as a result of ignorance! However, I hope I have persuaded some of you at least that by studying colour, its origins in Physics and Chemistry and its role in Life, we can enjoy Science.

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