Cryo-EPR of light-driven NADPH:protochlorophyllide oxidoreductase

Abstract

Protochlorophyllide (Pchlide) is the substrate of the light-driven reaction catalysed by the protochlorophyllide oxidoreductase (POR) enzyme. This reaction is a crucial step for chlorophyll biosynthesis in plants, it plays an important role in plant development and it has been suggested to also play a photoprotective role in plant cells. The POR reaction is one of the very few enzymatic reactions activated by light. The fact that the reaction can be triggered with a single laser pulse makes it a unique model to study enzyme catalysis. A crystal structure of POR is to date unavailable and this lack has thwarted a full elucidation of the reaction mechanism as well as the determination of the protein ternary structure and the exact active state geometry. The substrate, Pchlide, has unique excited-state properties and the decay kinetics including what was presumed to be a long-lived triplet state. We have used time-resolved absorption and electron paramagnetic resonance (EPR) spectroscopy at cryogenic temperatures to provide direct evidence of the triplet state of Pchlide and the characterisation of its EPR signature. We have found that the triplet state of Pchlide reacts with solvated oxygen, and that POR-binding reduces the triplet lifetime, supporting the theory of POR playing a photoprotective role. We were able to stabilise and characterise a highly transient reaction intermediate by means of cryo-trapping as well as identifying changes in its structure, a matter where there is currently no clear consensus. By studying substrate analogues we have been able to expand current knowledge on which and how different regions of Pchlide are important for the reaction photochemistry. The work in this thesis opens up this crucial biological system to a whole new range of experimental techniques that can prove instrumental in elucidating the still unclear reaction mechanism of light-driven POR.

Date of Award	1 Aug 2019
Original language	English
Awarding Institution	The University of Manchester
Supervisor	David Collison (Supervisor), Floriana Tuna (Supervisor), Nigel Scrutton (Supervisor) & Alistair Fielding (Supervisor)