Insight into multifunctional cytochrome P450 enzyme complex formation : a QCM study
Creators: name: Yeung, Chun In
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: Cytochrome P450 (P450) enzymes are haem-containing proteins that catalyse the oxidation reaction of exogeneous and endogeneous organic compounds. The cytochrome P450 catalytic cycle requires an input of substrate, oxygen and two electrons, which are delivered by redox partner protein(s). The cytochrome P450 enzymes are divided into two main classes, depending on the nature of redox partner protein(s) and their mode of interactions with the cytochrome P450 enzymes. Despite intensive research on the cytochrome P450 enzymes, very little detail is known about each class in terms of the protein-protein interactions between the cytochrome P450 enzyme and their redox partner protein(s). The formation of these protein complexes, which are necessary for their physiological functions, is far from being completely understood. This thesis aimed to study the formation of cytochrome P450 enzyme protein complexes in vitro. In particular, an exploration of the protein-protein interactions between the cytochrome P450 enzymes and the associated redox partner protein(s) has been undertaken. Three cytochrome P450 enzymes, CYP199A4, P450scc and P450c17, were used as representatives of a Class I bacterial cytochrome P450, a Class I mitochondrial cytochrome P450 and a Class II microsomal cytochrome P450 enzymes, respectively. The physiological environment was recreated by using biomimetic surfaces, either self assemble monolayers (SAM) or various compositions of lipid membranes, to study these cytochrome P450 enzymes. The P450-surface interactions as well as the protein-protein interactions with the electron donating redox partner protein(s) were monitored using a Quartz Crystal Microbalance with dissipation monitoring (QCM-D). This surface-based technique detects mass changes as the proteins are introduced, in solution, using a flow chamber via the change in frequency of an oscillating, gold coated, quartz crystal sensor, and provide an assessment of the amount of protein bound. In the configuration used here, simultaneous measurement of the structural properties of the deposited layers are provided via the decay features in the oscillation wave due to variations in the properties on the adsorbed layer(s). The real-time detection of both mass and structural changes using a QCM-D, has advantages over other techniques, and is a valuable approach to study biomolecules in vitro. A Class I bacterial cytochrome P450 (CYP199A4), a Class I mitochondrial cytochrome P450(P450scc) and a Class II microsomal cytochrome P450 (P450c17) enzymes were studied in details in Chapter 3, Chapter 4 and Chapter 5, respectively. The investigation on these three different types of cytochrome P450 enzymes led to a discovery of a common thread in which the nature of the underlying SAM or membrane layer was important for the deposition of cytochrome P450 enzymes. The consequent interactions with the cytochrome P450 redox partner protein(s) allowed the formation of specific protein complexes on the biomimetic surfaces. The binding and organisation of different classes of cytochrome P450 protein complexes could be regulated by the protein redox states and the specificity of the redox partner protein(s). These specific protein-protein interactions were identified and presented as the unique QCM-D binding 'fingerprints' for each cytochrome P450 system. The research described in this thesis has successfully demonstrated the interactions between cytochrome P450 enzymes and their redox partner protein(s) on biomimetic surfaces in vitro. The use of QCM-D enabled monitoring of the protein-surface and protein-protein interactions required for catalytic functions in real-time. This work presented here can be further extended to study the catalytic properties of these cytochrome P450 enzymes and their structure-function relationships, by using biomimetic environment developed in the current work.