'The Pigments of Life' 7th Schrdinger-Lecture Trinity College, Dublin, October 25, 2001 Bernhard Krutler

Institut fr Organische Chemie, Leopold-Franzens-Universitt Innsbruck, Austria

Abstract. Coloured compounds are an essential attribute of the developed forms of life. Indeed, the most visual sign of life on earth is due to the chlorophylls, that give the plants their green colour and that belong to the class of the porphinoid pigments. The natural porphinoids, called the "pigments of life", comprise metal free tetrapyrroles and metal complexes, such as the iron complex heme, the magnesium complex chlorophyll, the nickel complex coenzyme F430 and the corrinoid cobalt complex coenzyme B12. The natural porphinoids play central roles in all spheres of life, e.g. as cofactors for transport of oxygen, nitric oxide and electrons, for collecting and transforming solar energy, and as catalysts in biosynthesis. How did the porphinoid structures develop in nature ? How do their unique properties arise from the interactions between the metal centers and the tetrapyrrolic ligands ? To which particular chemical reactivities and modes of action do the porphinoid compounds owe their specific biological functions ? These questions may pave the way to insights into how life developed on earth. They also may open the prospect, that the properties of the porphinoid pigments might not only be beneficial for the living nature, but could be exploited profitably otherwise, e.g. by the use of porphinoids as catalysts for laboratory synthesis, for the storage and conversion of energy and for medical purposes. Ladies and Gentlemen, I am very much honoured to give this years' Schrdinger Lecture. It reminds us of Erwin Schrdinger, a great Austrian scientist, in the 40th year after his death. Needless to say, the famous Schrdinger equation is mentioned and used on this world many times every day, not only in connection with problems in physics, but also in chemistry. As an Austrian chemist, I am very pleased to be able to report to you on the "Pigments of Life", the compounds that bring colour to life (Sir Alan Battersby has used this description earlier). As you will see, my lecture will deal with natural pigments that are essential for life on earth, and thus it will deal with the material side of the colours in life. Schrdinger has made very important early analytical contributions to the "Farbenlehre", the physical description of colours and on how we perceive them.

2 Introduction According to the Holy Bible, God made mankind on the sixth day of creation. Here I show you Chagall's view of the creation of Adam. It appears as if Adam still is quite weak at this moment. To emphasis this, Chagall chose Adam to be only very faintly coloured. But he already has some colour, which is meant to indicate to us that life is already in him. What does colour have to do with human life ? With life, in a more general sense ? Charles Darwin has dealt with this question in a more thorough way, by stating in his book "Descent of Man and Selection in Relation to Sex" (London 1871): "Hardly any colour is finer than that of arterial blood; but there is no reason to suppose that the colour in itself is an advantage; and though it adds to the beauty of the maidens cheek, no one will pretend that it has been acquired for that purpose". As you can see, Darwin is sceptical, that colour itself is of an evolutionary advantage to mankind. The Iron-porphyrin Heme Clearly an important source of the colour of the human body is the blood. There it is the hemoglobin, the red coloured enzyme that binds and transports molecular oxygen. Let us look at a model of the oxygen binding and transporting enzyme myoglobin, the smaller analogue of hemoglobin in the muscles. The model shown here was developed by X-ray analysis of crystalline myoglobin. This enzyme consists of two major moieties, the colourless protein part of this enzyme (the apoenzyme) and the cofactor, which actually is a red coloured pigment, the iron porphyrin heme. Let us focus now on this coloured compound (see Figure 1)[1]: CH3 H3C N Fe H3C N N CH3 N

HO2C Figure 1: Structural formula of heme

CO2H

Shown here on the left side is the structural formula of heme, an iron complex of protoporphyrin, as this tetrapyrrolic ligand is called. This porphinoid iron complex was synthesised in the laboratory of Hans Fischer, mainly to prove or derive its molecular structure. On the right side of this slide you see a projection of the structure of hemin is shown in a view nearly vertical onto the porphyrin plane, as determined by X-ray analysis. Let us go back to the enzyme myoglobin: we focus now on the section of the crystal

3 structure of myoglobin, which is occupied by the porphinoid cofactor heme. This model is from the structure of myoglobin that already carries an oxygen molecule. The heme cofactor is seen from the side: the view is in the porphyrin plane. An oxygen molecule is bound endon to the iron center of the bound heme. This binding mode was predicted by Pauling, based on the unique electronic properties of the heme and of the oxygen molecule. So heme is having the right chemical properties for binding of molecular oxygen. However, quite disturbingly, when heme is exposed to molecular oxygen in free solution, it is oxidized to hemin irreversibly. This is detrimental for its function, as hemin is not able to bind molecular oxygen to any significant extent. How does the protein surrounding in myoglobin or hemoglobin protect the heme from becoming oxidised ? The answer turned out to be quite simple: it was experimentally provided by J. Collman, in whose labs the picket-fence porphyrins were developed and synthesised. A model structure of these entirely artificial relatives of heme is shown on this slide, actually already binding an oxygen molecule at the iron center. As suggested by Collman, the bulky fence protects this oxygen carrying analogue of heme from becoming oxidized. A major role of the protein part in myoglobin therefore simply concerns a similar protection of the heme cofactor. Indeed, picket-fence porphyrin reversibly binds molecular oxygen: it can be considered to be a minihemoglobin. The capacity of heme to interact with molecular oxygen and to bind it, has been exploited by nature in a second class of enzymes, the cytochromes P450. These enzymes perform a unique kind of chemical catalysis. Here I show you a model of such a cytochrome P450 from the microorganism Pseudomonas putida. Similar enzymes also exist and do their important work in your bodies. The cytochromes P450 are important catalysts for the oxygenation of a range of substrates. Shown here is the case of a so called camphor hydroxylase, an enzyme that puts a hydroxy group into the camphor molecule. It introduces this new functionality in a position that would be exceedingly difficult to hydroxylate by a conventional chemical reaction. As can be gathered from a three dimensional model of the enzyme, obtained from x-ray analysis, a colourless protein cage provides room for the cofactor (heme) and the substrate (camphor). Indeed, only when the substrate is bound near the heme cofactor (the same heme as in hemoglobin) the catalytic process is triggered. This strict control is important, as the reaction involves chemically highly aggressive oxygen species. When we look at the active site of the enzyme more closely (with the heme cofactor), the substrate molecule is seen to sit above the iron center of the heme cofactor. The camphor molecule is thus at a place, where an oxygen atom bound to the iron center can directly interact with it at the proper position and abstract a hydrogen atom there. As mentioned, the cytochromes P450 achieve chemically very difficult reactions. Here again, the electronic properties of heme cofactor allow for its unique role in binding and activating molecular oxygen. Clearly, it is the electronic properties of heme which are exploited in these enzymes. At the same time the electronic build-up of heme gives it a red colour, which presumably has no physiological consequence. The Cobalt Corrin Coenzyme B12 Let us now turn to another red pigment in your bodies, that is much less visible than heme. The red compound that I show you here as beautiful red crystals [2], was searched for in the first half of last century as a cure for pernicious anemia. It was named vitamin B12 and turned

4 out to be a cobalt complex. Vitamin B12 resisted structural analysis by the established chemical means. However, with the help of crystalline vitamin B12, its three dimensional structure could be solved by X-ray analysis in the laboratory of D. C. Hodgkin. Based on the crystal structure, the structural formula (shown in Figure 2) was eventually obtained. H2NOC H2NOC H3C H3C H H2NOC CH3 CONH2 CH3 N N CH3 CH3 H3C CH3 O CONH2 CH3 CH3 CONH2

N N

R Co +

HN H3C H O P O O

N N O OH

HO

Figure 2: Structural formulae of vitamin B12 derivatives: vitamin B12 (R = cyanide), coenzyme B12 (R = 5'-deoxyadenosyl), methylcobalamin (R = methyl). As you can see, this cobalt complex again is a tetrapyrrole derivative, and is thus related to the heme molecule. The cobalt ion is bound by a so called corrin ligand. This tetrapyrrole ligand is much more complex than the porphyrin ligand of the heme. One of the substituents at the periphery of the corrin ligand in vitamin B12 is a unique appendage, a nucleotide function, that coordinates back to the cobalt center. One of the important reactions catalyzed by B12 is the complex rearrangement of the carbon skeleton of methylmalonyl-coenzyme A to succinyl coenzyme A. This again is a reaction, which is very difficult to achieve by chemical means. How could such a reaction be catalyzed by the B12 molecule ? In fact, a new type of B12 was discovered to act as cofactor in this reaction. This B12-cofactor was called coenzyme B12. Its structure again was elucidated by the help of an x-ray analysis. Coenzyme B12 was found to have the same corrin-nucleotide moiety as vitamin B12, but to carry an adenosyl group at the cobalt center (see Figure 2). In vitamin B12 a cyanide group is bound at the same position. Vitamin B12 actually is an artefact

5 of the isolation procedure and has no direct physiological role in your bodies. Coenzyme B12 is an organometallic compound, a unique situation for a cofactor. Indeed, the biological roles of coenzyme B12 are intimately connected with the CoC-bond. How then does coenzyme B12 catalyse the mentioned complicated rearrangement reaction ? Very recently the structure of methylmalonyl CoA mutase was solved by X-ray analysis. This is the enzyme that catalyses the before mentioned rearrangement of methyl malonyl coenzyme A to succinyl coenzyme A. The enzyme, whose crystal structure was solved, actually is not the human enzyme, but a bacterial enzyme from Propionibacterium shermanii. In this enzyme, a colourless protein tightly binds the red cofactor. The protein embraces the cofactor tightly and provides a kind of a Procrustes' bed for it. It is one of the main jobs of methylmalonyl-CoA mutase to put strain on the bound cofactor and to induce the fragmentation of its rather weak CoC bond. One of the fragments is a highly reactive radical, which then attacks the substrate to induce it to undergo the rearrangement reaction. A second important reaction in your body, that is catalyzed by B12, is the synthesis of the proteinogenic amino acid methionine by methylation of homocysteine. The methyl group used is originating in the methyl group of methyl-tetrahydrofolate. How can B12 achieve this enzymatic transformation ? The enzyme involved here, methionine synthase, is a complicated, four domain species, one of which binds the B12 cofactor. In fact, it is not coenzyme B12, that is used here, but another organometallic B12 derivative, carrying a methyl group at the cobalt center and called methylcobalamin. The cobalt-bound methyl group can be easily transferred and can be used in a range of biological methylation reactions. Both, methylcobalamin and coenzyme B12 are highly light sensitive compounds. They both are quite rapidly decomposed by light. Clearly again, it is the electronic properties of these coloured organometallic B12-cofactors, that are relevant for their functions for life and not the colour itself. The Magnesium Chlorin Chlorophyll With that let us turn to the pigment of life that is the most visible in nature, to the chlorophylls. Here in Ireland, I am sure, one does not need to point out the abundance of chlorophyll. The chlorophylls are the most essential pigments in green plants and other photoorganisms: they help to collect sun light and then to transform it into chemically useful equivalents of energy. In the green plant, the biological energy currency adenosine triphosphate is provided this way, as well as the reduction agent NADPH, the hydride of the nicotineamide-adenine-dinucleotide-phosphate. These two then help drive the Calvin cycle, which leads to the fixation of carbon dioxide. The green pigments of the plants are the chlorophylls a and b. Let us look here at the structural formula of chlorophyll a (see Figure 3). It is the magnesium complex of the reduced porphyrin pheophytin. The green colour resides in the tetrapyrrolic ligand. Besides its tetrapyrrolic magnesium coordinating macrocycle, chlorophyll a has a lipophilic substituent, that helps anchor this photo-reactive molecule into its biological matrices. You will not be surprised to hear that the metabolism of the chlorophylls presumably is the most visible sign of life on earth. Chlorophyll metabolism can actually be seen from outer space. Shown in this figure, are four representative pictures of Europe taken from a satellite in the course of the year 1995 and colour coded according to the so called vegetation index. One can see, how the chlorophyll molecules are synthesised in the first half of the year and

6 how they disappear in the second. Of course, as we can see also, Ireland is green all year round. A large amount of knowledge has accumulated on the biosynthesis of the chlorophylls. In contrast, the disappearance of the green plant pigments remained enigmatic until very recently and seemed to occur without leaving a trace of them.

CH3 H3C

Mg
N
H3C

N
CH3

CH3 C O C
O

O OCH3 CH3 CH3 CH3 O CH3

O O OH

CH3

O
H3C H

NH NH

HN HN
CH3

H3C H

C
OH

C O
OH

Figure 3: Structural formulae of chlorophyll a (above) and of the colourless and nonfluorescent chlorophyll catabolite from degreened cotyledons of oilseed rape (Bn-NCC-1, bottom)

The latter seemed highly curious, since it has been estimated that annual turn-over of the chlorophylls involves around one-thousand million tonnes. The knowledge on the fate of chlorophyll was not only of an academic interest, but also of an economic and ecological one. When you look out of the window now, you will notice the change of the colours of the leaves. This also occurs in Austria, of course. Why should nature be interested at all in degrading the beautiful chlorophyll molecules ? What can easily be seen out there in nature to happen on a large scale, can also be studied on a smaller laboratory scale. Shown here are representative phases in the first 25 days after seeding of oilseed rape, an important agricultural plant. The two green cotyledons of this dicot develop pairwise and degreen under natural growth conditions, when the second generation of leaves has matured. In these yellow leaves, chlorophyll has disappeared and the remaining yellow colour presumably is due to the carotenoids present in the leaf. In collaboration with Prof. Philippe Matile of the Botanical Institute of the University of Zrich we were able to study chlorophyll breakdown in such degreening cotyledons of oilseed rape. The main remains of the chlorophylls in the yellow leaves of oilseed rape is a colourless compound, whose chemical formula is shown here (Figure 3)[3]. Similar compound have been isolated from degreened leaves from a range of higher plants, such as barley, pepperoni, spinach. They all show a similar basic chemical build-up. Clearly, the disappearance of the chlorophylls is an orderly process in the senescent plant and not an irregular decay. When comparing the structural formulae of the chlorophyll molecules and that of the colourless (and nonfluorescent) chlorophyll catabolite, one may now ask two obvious questions: How do those structural changes occur in the senescing leaf ? and For what possible purpose does chlorophyll breakdown occur at all ? Let us first deal with the first type of question. We know now, that in the ordered senescence process, such colourless catabolites are produced in five steps from chlorophyll a, most of which are highly regulated enzymatic processes. It all starts with the loss of the phytol chain followed by the removal of the magnesium ion. At this stage, the tetrapyrrolic remains of the chlorophyll molecule still has essentially kept the green colour. In three further steps the green pigment is rapidly cut up enzymatically to end up in these colourless remains. The colourless chlorophyll catabolites are still tetrapyrroles: The nitrogen contained in the chlorophyll molecule is still present in these degradation products. Clearly, our results do not support the hypothesis, that chlorophyll breakdown has as its goal the recuperation of the four nitrogens of the green plant pigments. For what purpose could chlorophyll breakdown occur then ? This question is not settled, but two main consequences of chlorophyll breakdown may be very helpful to the plant. For once, the rapid destruction of the green chromophore of the chlorophyll molecules eliminates their potential phototoxic effects: In the presence of oxygen, photoexcited chlorophylls produce oxygen species toxic to the cell, such as singlet oxygen. In addition, the removal of the chlorophyll molecules helps present the chlorophyll binding proteins to proteases. It is the abundant nitrogen in the proteins that the plant is after. Clearly, the colour of the green plant pigments is intimately connected with their function as photocatalysts. The photoactivity of the chlorophylls is beneficial to the plant, when these green pigments are bound in the intact light harvesting units and photoreaction centers. On

8 the other hand, the chlorophylls and related porphyrins may be very photo-toxic, when not well controlled and organized. Concluding Remarks I have reported to you on some important pigments of life, on the B12 derivatives, on heme and some of its enzymes and on the chlorophylls. All of these pigments are so called tetrapyrroles with a common origin. They are all produced in nature from the porphyrinogen, uroporphyrinogen III. From this cyclic tetrapyrrole also other important porphinoid metal complexes are derived naturally, which I have not mentioned in more detail. The porphinoid natural products are the most visual and probably ubiquitous pigments in the living nature other pigments do also have important functions, of course: I may mention the carotenoids, the flavonoids and the anthocyans. The porphyrins shown here are probably unique due to their high abundance in all spheres of life, their visibility and the wealth of their biological roles. With this, Ladies and Gentlemen, let me come to an end with my lecture. I would like to thank you for your kind attention. I would also like to thank my coworkers for their important contributions. Shown on this slide, taken in fall 2000, you see Michael Oberhuber, Joachim Berghold and Sigrid Ostermann, graduate students currently working in my group in the areas of chlorophyll breakdown and organometallic B12 chemistry. I am also particularly grateful to Philippe Matile and his group for a wonderful interdisciplinary collaboration. I would like to thank all of my coworkers and collaborators for their fruitful interactions and the Austrian National Science Foundation (FWF) for financial support of our research.

Selected References [1] B. Krutler, Chimia 41, 277 (1987). [2] B. Krutler, D. Arigoni, B. T. Golding (Eds.) Vitamin B12 and B12-Proteins, Verlag Chemie, Weinheim (1998). [3] B. Krutler, P. Matile, Acc. Chem. Res. 32, 35-43 (1999).