The study of the reaction of carbon monoxide with Pseudomonas putida cytochrome P450cam
Creators: name: Albon, Simon Piers
Publisher: name: University of British Columbia
Year: 2010

description: Interest remains very high in the class of thiolate-bound heme-containing monooxygenase enzymes called the cytochromes P450. These enzymes generally activate molecular dioxygen for selective hydroxylation of organic substrates, such as saturated hydrocarbons (RH), in accordance with Reaction (1). RH + O₂ + 2e⁻ + 2H⁺ ￫ ROH + H₂O (1) Development of protein-free model catalysts that mimic P450 reactivity are of great industrial importance and require, in part, an understanding of the reactivity of the native hemoprotein towards small gas molecules such as carbon monoxide (CO) and dioxygen (02). The work in this thesis describes the reaction of carbon monoxide with the ferrous state of substrate-bound, native and protohemin-reconstituted Pseudomonas putida cytochrome P450cam (Reaction (2)). [equation not included] A method is described for the growth of P. putida, strain PpG 786, and the subsequent isolation and purification of native cytochrome P450cam and associated electron transport proteins, putidaredoxin and putidaredoxin reductase; only purified cytochrome P450cam and putidaredoxin were successfully obtained by this method. The native enzyme has been reconstituted with protohemin by a method general for the insertion of hemins modified in the 2- and 4- positions. Meso-, deutero-, diacetyldeutero-, and dibromodeuterohemins were synthesized but were not used for reconstituting the native cytochrome. Spectral analyses were performed on the substituted hemins, the native cytochrome and related electron transport proteins and the protohemin-reconstituted enzyme; the data agreed well with literature values. Kinetic constants for the forward CO-association and reverse dissociation reactions (k[sub on] and k[sub off], respectively; see Reaction (2)) were determined by flash photolysis; experiments were run under conditions in which the CO ligand was in substantial excess and all systems showed pseudo first-order dependence on hemoprotein concentration. The flash photolysis unit described below was tested and developed with the myoglobin-CO system and then used to study the substrate-bound native and protohemin-reconstituted P450cam-CO systems. The second-order rate constants for CO-binding to ferrous myoglobin and native substrate-bound P450cam were in excellent agreement with published values and reconstitution of the native P4 50cam enzyme had little effect on the k[sub on] value for CO-binding. The first-order rate constants for the C0- dissociation reaction were determined also from the flash photolysis studies but were considered unreliable when compared with published literature data. A k[sub off] value for the native P450cam system, in good agreement with the literature, was calculated from the equilibrium constant, Kco, derived by spectrophotometric analysis of Reaction (2); Kco values in reasonable agreement with literature data were obtained but indicated an interesting dependence on the concentration of the dithionite reducing agent. This thesis begins with an extensive literature review on the subject and ends with some general conclusions on this work and some recommendations for future work.