Cytochrome P450cin. An Investigation into the Substrate-Based Determinants of Reactivity

Cytochromes P450 constitute a superfamily of approximately 7000 oxidative hemoproteins found across all domains of life. Despite their importance in such roles as the biosynthesis of steroid hormones and metabolism of xenobiotics, the physical and chemical properties of P450s are not always well understood. As a consequence of the difficulty in isolating these enzymes without denaturation, most investigations of P450s have examined only a small handful of robust “representative enzymes”. The greater the cross-section of well-characterised enzymes presented in the literature, the more we can deduce the commonalities and differences between P450s with confidence. The current knowledge of P450s, as well as the significance of the discovery of cytochrome P450cin, is discussed in chapter 1. The recently-discovered P450cin isolated from the soil bacterium Citrobacter braakii, is of interest not only because of its relative stability (hence making it a convenient subject of study as a “representative enzyme”), but also because it introduces chirality in an achiral molecule, cineole (C10H18O). The importance of chirality in biological molecules is well recognised and asymmetric induction is now a vital tool in, amongst other things, the development of pharmaceuticals. This thesis presents further characterisation of cytochrome P450cin and is primarily concerned with the substrate-based mechanisms of reactivity. In order to do this, a series of probes derivatives of cineole, camphor, and bicyclo[2.2.2]octane were synthesised. The syntheses of these molecules is described in chapter 1. The capacity of P450cin to oxidise these probes was then tested in vitro. Where relevant, the P450cin-catalysed oxidation of these probes was characterised further by measuring dissociation constants, hydrogen peroxide production, rate of consumption of NADPH and product formation relative to the quantity of available NADPH. The capacity of P450cin to carry out reactions other than hydroxylations, especially with regard to the potential reactivity of a P450cin-peroxy complex, was investigated in chapter 3. The capacity for P450cin to carry out oxidative diol cleavage reactions was also investigated, as were the substrate-based determinants of selectivity. The results reported in this thesis indicate that P450cin exhibits predilection for hydroxylation over other possible oxidative processes (C-C bond cleavage, peroxide-mediated processes etc.) has been observed, although peroxide-mediated processes can occur under certain conditions. Furthermore, regioselective and enantioselective oxidations were largely limited to substrates containing an endocyclic ethereal oxygen. It was intriguing that one probe, an α-fluorinated ketone, unexpectedly inhibited the enzyme. Preliminary results suggest that this deactivation is dependent on the locality of the ketone with respect to the heme complex of the enzyme. It was observed that camphane (a deoxygenated substrate comprised only of carbon and hydrogen) was oxidised by P450cin to the ketones camphor and epicamphor, but the intermediate alcohols were not observed. This led to speculation that the ethereal oxygen of the natural substrate may be important not only for determining the regioselectivity of oxidation, but also in the efficiency of oxidation, and that such deoxygenated molecules may be poorer substrates than the immediate oxidative products. Further experimentation presented in chapter 4 has debunked this hypothesis