Biophysical and structural studies of chlorophyll biosynthetic enzymes magnesium chelatase and protochlorophyllide reductase

Photosynthetic organisms must generate chlorophyll, the light capturing molecule integral for photosynthesis, whilst also regulating production to adapt to environmental conditions. Chlorophyll is so fundamental for photosynthesis that it is important to understand its biosynthetic pathway; consequently, this sequence of reactions has been studied extensively for several decades, culminating in the engineering of the heterotroph E. coli to produce chlorophyll. Two key regulatory points in the pathway are i) at the branchpoint between haem and chlorophyll biosynthesis, where a magnesium ion is inserted into the porphyrin ring, catalysed by the multi-subunit enzyme magnesium chelatase (MgCH); and ii) the light-dependent reduction of the C17-C18 double bond, catalysed by protochlorophyllide oxidoreductase (POR). Despite the extensive work performed to biochemically describe these enzymes, the full reaction mechanisms are still unknown, and the limited amount of structural information for either enzyme has hindered complete characterisation.
The work reported in this thesis has used a range of biochemical, biophysical and structural techniques to study these biologically important and mechanistically unique enzymes: microscale thermophoresis, kinetic analysis and cross-linking mass spectrometry revealed that the driving force behind chelation, the ATPase activity of the AAA+ ChlI subunit, is linked to the chelating ChlH protein via the regulatory ChlD subunit; X-ray crystallography-guided mutagenesis determined the porphyrin binding site in ChlH; (cryo)-electron microscopy was used to initiate structural investigations into the quaternary organisation of MgCH and POR; and 2-dimensional electronic spectroscopy was applied to the light-initiated reaction catalysed by POR, the first use of this complex technique on an enzyme, potentially revealing a novel intermediate in the reaction that is formed on the femtosecond time-scale. The work presented in this thesis aims to develop our biochemical understanding of MgCH and POR, and lay the foundations for further structural and mechanistic studies of these interesting enzymes of chlorophyll biosynthesis.