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Studies on carbon monoxide and dioxygen binding to cytochrome P-450cam

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Title: Studies on carbon monoxide and dioxygen binding to cytochrome P-450cam
Author: Rajapakse, Nimal
Degree Master of Science - MSc
Program Chemistry
Copyright Date: 1984
Subject Keywords Cytochrome P-450
Abstract: Interest has remained very intense during the last two decades on the heme-containing monooxygenase system, cytochrome P-450. The P-450 hemoproteins are widely distributed in nature and engage in oxygenation of a wide variety of substrates according to the reaction, R-H + 0₂ + 2H⁺+ 2e⁻ → R-OH + H₂0 where R-H represents an unactivated carbon-hydrogen bond. Investigations on binding of small gas molecules such as CO and 0₂ to the P-450 enzymes are important not only in understanding various aspects of monooxygenation but also in developing protein-free model systems that can mimic the catalytic properties of P-450. This thesis describes gas binding studies carried out on cytochrome P-450cam. A procedure is given for growing the bacterium Pseudomonas putida strain 786 from which soluble, camphor hydroxylating P-450 enzyme is isolated and purified. The binding of CO to the stoichiometrically reduced substrate-free enzyme at different temperatures was studied using a standard spectrophotometric procedure. From these experimental data, the thermodynamic parameters ΔH⁰ and ΔS⁰ were calculated for the reaction, (P-450)Fe(II) + CO ⇌ (P-450)Fe(11)-C0 . Attempts to determine such thermodynamic parameters for the binding of dioxygen to the substrate-bound P-450 enzyme were not successful. On comparison of the determined thermodynamic parameters for the substrate-free system with the literature values for substrate-bound enzyme, hemoglobin, myoglobin and P-450 model systems, it is concluded that the substrate molecule was bonded in the immediate vicinity of the active-site thereby lowering the CO affinity to the substrate-bound system.
URI: http://hdl.handle.net/2429/24901
Series/Report no. UBC Retrospective Theses Digitization Project [http://www.library.ubc.ca/archives/retro_theses/]
Scholarly Level: Graduate

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