Photoenzymes use light to initiate biochemical reactions. Although rarely found in nature, their study has advanced understanding of how light energy can be harnessed to facilitate enzyme catalysis, which is also of importance to the design and engineering of man-made photocatalysts. Natural photoenzymes can be assigned to one of two families, based broadly on the nature of the light-sensing chromophores used, those being chlorophyll-like tetrapyrroles or flavins. In all cases, light absorption leads to excited state electron transfer, which in turn initiates photocatalysis. Photocatalysis by protochlorophyllide oxidoreductase (POR) â one of only a few natural light-dependent enzymes - is responsible for the reduction of protochlorophyllide to chlorophyllide in chlorophyll biosynthesis. Photocatalysis by POR not only regulates biosynthesis of the most abundant pigment on Earth, it is also a âmaster switchâ in photomorphogenesis in early plant development. Upon illumination, POR promotes chlorophyll production, plastid membranes are transformed, and the photosynthetic apparatus is established. Given these remarkable, light-induced pigment and morphological changes, the POR-catalysed reaction has been studied extensively from catalytic, physiological and plant development perspectives, highlighting vital, and multiple, cellular roles of this intriguing enzyme. The aims of this thesis were to develop the current understanding of the POR reaction mechanism by investigating different structural models for the POR ternary enzyme-substrate complex and how these may impact the active site residues that are potentially involved in the reaction chemistry. A newly designed model built from existing computational models was hypothesised to provide a feasible structural model that could be used to rationalise key binding residues, potential binding positions of Pchlide and understand the role of key residues involved in POR catalysis. Three key active site residues were explored in detail and their potential involvement in the mechanism is discussed. One of these key residues is a tyrosine which is commonly referred to in the literature as being the proton donor of the light-activated reduction reaction. Mutagenesis and ultra-fast laser spectroscopy studies were carried out which suggest otherwise, since no significant change in reaction kinetics was measured for fluorinated-tyrosine variants. The key role of this tyrosine is highlighted as facilitating successful binding of the ternary complex, which is critical for enzyme efficiency. Recently, a cysteine residue was suggested as an alternative proton donor, so the role of this residue was investigated in this work by mutating to alanine. The results for the C226A variant demonstrate changes in the absorbance spectrum measured during the reduction reaction, an increase in rate of hydride transfer and a decrease in rate of proton transfer. Taken together this indicates the likely involvement of the residue in either one or both of these transfer steps. Finally, a glutamine was highlighted as a residue of interest in the newly designed structural model and this residue was also mutated to alanine. A significant detrimental effect to the binding affinity of the ternary complex was measured with the Q248A variant, and an unexpected increase in rate of both hydride and proton transfer was also noted. A putative alternative mechanism was explored, involving tautomerisation of the Pchlide substrate prior to catalysis. The oligomerisation of POR upon binding the Pchlide substrate was investigated and a range of detergents and natural lipids were screened as a means of producing a heterogeneous sample of the ternary complex. The work in this thesis builds on up-to-date knowledge of the mechanism of the POR reduction reaction and provides a foundation for future research in order to develop our understanding of photoenzymes.
- Spectroscopy
- Enzymology
- Protochlorophyllide Oxidoreductase
- Photoenzymes
- Bio-catalysis
Investigating the reaction mechanism of Protochlorophyllide Oxidoreductase
Taylor, A. (Author). 1 Aug 2023
Student thesis: Phd