Nature uses sunlight either as energy source or as information carrier. Photoreception is achieved by two groups of specialised proteins: photo-enzymes that catalyse photoreactions and photosensors that initiate physiological functions. In the present work mechanistic studies were conducted on one representative of each group by using site-directed mutagenesis as well as stationary and time-resolved spectroscopy. The photo-enzyme NADPH:Protochlorophyllide Oxidoreductase (POR) catalyses the light-dependent C17-C18 double bond reduction of protochlorophyllide, including a hydride and a proton transfer, to produce chlorophyllide, the immediate precursor of chlorophyll. POR provides a unique opportunity to study the hydride transfer mechanism in detail. Three distinct intermediates, prior to product formation, were observed that were interpreted as electron and proton-coupled electron transfer reactions from NADPH indicating a sequential hydride transfer mechanism. An active-site mutant, POR-C226S, yields distinct different intermediates compared to POR wild type but ends in the same chlorophyllide stereoisomer most likely due to an altered protochlorophyllide binding. This work provides the first direct observation of a stepwise hydride transfer mechanism in a biological system. Cryptochromes (CRY) are so far defined as flavoprotein blue-light photosensors that regulate the circadian clock throughout nature and are suggested as the candidate magnetoreceptor in animals. Animal CRY are subdivided into two classes of proteins: the light-responsive Type I (invertebrates) and the light-independent Type II (mainly vertebrates). The molecular basis of their different roles in the circadian clock is still unknown. Animal Type I CRY are suggested to undergo conformational changes - required for induction of subsequent signalling cascades - induced by the change in the FAD redox state due to light absorption. The study shows that in contrast to Type I animal Type II CRY do not bind tightly FAD as a cofactor due to the lack of structural features and therefore provide the molecular basis for their different biological roles ruling out a direct photomagnetoreceptor function. Further, detailed studies on a fruit fly (Dm)CRY reveal that it does not undergo a photocycle as FAD release and Trp decomposition were observed. Thus, it is suggested that light is a negative regulator of DmCRY stability linking the initial photochemistry to subsequent dark processes leading to signal transduction on a molecular level.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:764593 |
| Date | January 2018 |
| Creators | Archipowa, Nataliya |
| Contributors | Graham, Darren ; Scrutton, Nigel |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | https://www.research.manchester.ac.uk/portal/en/theses/mechanistic-studies-on-the-lightdependent-nadphprotochlorophyllide-oxidoreductase-and-animal-cryptochromes(667672e1-d037-4135-b4d2-71a98331834f).html |
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