Enzyme models of chloroperoxidase and catalase

As early as the 17th century it was recognized that one component in air is essential for life. This extremely simple molecule later known as dioxygen or commonly oxygen was discovered by Joseph Priestley and Carl Wilhelm in 1771-72 by thermal decomposition of mercury oxide and potassium nitrate respectively. During the next three years Lavoisier worked on the characterization of the new element composing dioxygen. In 1775 he named it oxygen due to its presence in numerous acids (oxy = acid, gen = generate). Lavoisier also established the role of dioxygen in the respiratory process. The fundamental equation dioxygen=life was formulated for aerobic systems. Nearly two centuries later, in 1929, the electronic structure of dioxygen was solved by Linus Pauling using the theory of molecular orbitals. Pauling could explain the paramagnetism of dioxygen (cM=0.994 T-1) as well as the interatomic distance (d=1.207) and the dissociation energy (Ediss=117.36 kcal mol-1) which were until then poorly understood by chemists. This state is defined as the triplet state and depicted in term of molecular orbital in figure 1. The pioneering theory of Pauling could also predict the two metastable states of dioxygen where the molecule becomes diamagnetic. Both states are known as D and S singlet states (figure 2) and differ from the orbitals filling: the 1Sy + state has the two electrons paired in the p* 2py orbital whereas the 1Dy has still on electron in each p* orbital but with reversed spin. The latter state has a life time of 45 Min. and an O-O bond distance of 1.223. These singlet states are important for the reactivity of dioxygen in particular in the generation of ozone and radical species. These radicals are responsible for the formation of hydrogen peroxide, a highly oxidizing substance discovered by Thenard in 1818. Accumulation of hydrogen peroxide in the cell is extremely dangerous as it destroys the protein structure. The relationship between oxygen, hydrogen peroxide and the respiratory processes has been more deeply understood during the 20th century. For example, myoglobin which binds oxygen was the first protein of which the structure was solved by X-Ray diffraction in 1960 [1]. This is showing the interests and efforts developed to establish how dioxygen is used by aerobic organisms. In the second part of the 20th century the progress of medicine and biology has provided a better understanding of the importance of dioxygen and led to the discovery of numerous enzymes which are related to dioxygen. Although dioxygen is the basic source of energy for aerobic systems, its consumption produces radicals such as superoxide ion (O2 -) in cells. This highly reactive radical is very toxic and implied in numerous pathologies. Fortunately a “dioxygen cycle” (figure 3) exists by which oxygen [3O2] can be activated or recycled such that toxicity problems are prevented. The close dependency of hydrogen peroxide (H2O2) and dioxygen reflects the complexity of the nature despite the use of very simple molecule for ensuring life. Moreover, hydrogen peroxide which is known as a cell killer due to its high oxidizing power is required as a substrate for many enzymes. A real paradox exists: hydrogen peroxide and the superoxide ion have to be present in living organisms but their concentration has to be controlled accurately so that they are only short living in cells. It was recognized early that metal containing species, in particular iron complexes, were involved in these processes. Rapidly the idea was accepted that a relatively simple unit is responsible for the transport and activation of dioxygen. Küster postulated a tetrapyrrolic structure [2] termed porphyrin. Its synthesis by Hans Fischer [3] established that its iron complex known as heme was able to bind dioxygen. Such heme units were shown to be essential for activating oxygen for oxidation reactions. There are now seven hemes known which differ from each other by the nature of the substituents on the b-pyrrole positions. The most common heme is heme b or hematin (figure 4). Several hundreds of heme b proteins are nowadays known which have different functions such as electron transfer, oxygen or nitric oxide transport or oxidation as shown in table 1. As all these proteins share exactly the same metal complex, it has been pointed out that the protein environment around heme b plays a decisive role in determining the function of the protein: 1) The amino acid residues maintain the heme unit bound to the protein 2) One amino acid residue coordinates axially to the iron in all heme proteins. The nature of this axial coordination is thought to be the most important factor which determines the enzyme’s activity. Therefore heme proteins are often classified according to the nature of the axial ligand (table 1). Finally the hydrophobic or polar character of the amino acid residues on the side opposite to the axial ligand (= distal side) tunes the specificity for a particular substrates and aids the formation of catalytic intermediates. The best known heme proteins are the globins, hemoglobin and myoglobin which are essential blood constituents. Peroxidases have also been the centre of intense studies as they were using hydrogen peroxide for oxidation in living organisms and have the same axial ligand, the nitrogen from histidine, as the globins. Less attention has been paid to Catalases which have a tyrosinate axial, similar to peroxidases. Finally heme-thiolate proteins are subject of special attention due to the unusual presence of a thiolate coordinating to the iron. Several heme models of the active site of the enzymes Chloroperoxidase and Catalase have been synthesized in order to study the influence of the axial ligand and peripherical meso heme substituents on the formation and characterization of catalytic intermediates. Each metal-free porphyrin was thoroughly characterized by NMR spectroscopy prior to the introduction of the iron atom to form the desired active site model. Each heme model was then characterized by UV-Vis, NMR, an EPR spectroscopy. Following results were observed: The attempts to obtain better models finally support the approach of bridged structures over tailed models. Contrary to the conclusion of Nakamura et al. with hydrogen bonded stabilized heme thiolate complex [122] we could not notice such an effect with the precursors 49 and 55. Nevertheless the results obtained for metal free tailed porphyrin have permit to establish that upon bridging the UV-Vis spectra are red shifted and that the conformation of the ortho amide substituents on the meso aryl groups is changed. For these reasons the future direction for having better binding constant is probably still to use an ortho amide function on the aryl moiety. But instead of having a bulky substituent directly linked to the carbonyl group of the amide this one should be anchored at a greater distance. For example the replacement of the tert-butyl group by a furan already improved the binding abilities of 11 [118].