Magnesium Chelatase: Insights into the first Step of Chlorophyll Biosynthesis

Abstract: The enzyme magnesium chelatase inserts magnesium into protoporphyrin IX (Proto) to produce to magnesium protoporphyrin IX, the first unique intermediate of the chlorophyll biosynthetic pathway. Magnesium chelatase is composed of three distinct proteins termed I (molecular weight ~40 kDa), D (~70 kDa) and H (~140 kDa). Defining the individual properties and structure of the magnesium chelatase components and their role in the reaction mechanism is important for a full understanding of the first step in magnesium tetrapyrrole biosynthesis. The three components of magnesium chelatase show significant conservation at the protein sequence level, which extends from bacteriochlorophyll synthesising purple non-sulfur bacteria and green sulfur bacteria to chlorophyll synthesising eukaryotes and cyanobacteria. In Paper I, eight mutants of the H gene (Xantha-f) from barley were characterised at the molecular level and provide explanations for the yellow phenotypes of germinating mutant seedlings. In this thesis magnesium chelatase from the photosynthetic bacterium Rhodobacter capsulatus has been used as a model system since much of the pioneering work has been conducted on this organism. Magnesium chelatase requires ATP to insert magnesium into Proto. There has been conflicting results as to the ATPase activity of the H subunit. In Paper II it was demonstrated that ATP hydrolysis can be attributed the I subunit and not the H. The unprecedented discovery of an iron-sulfur cluster in the H subunit of R. capsulatus is described in Paper III. The cysteine motif that coordinates this iron-sulfur cluster is only present in five other facultative proteobacteria and absent in all oxygenic or anaerobic species. The function of this cluster is yet to be established. In Paper IV the first insights into the structure of an H subunit is presented. Electron microscopy and single-particle reconstruction was used to solve the structure in the apo and substrate bound conformations at a resolution of 25 Å, and revealed a conformational change upon Proto binding. Limited proteolysis and construction of truncated H polypeptides provided supporting information to propose a cooperative substrate binding model. The binding of porphyrin to the H subunit was further investigated in Paper V using tryptophan fluorescence quenching to detect a high affinity porphyrin binding site in the nanomolar range. Alanine mutagenesis of the H subunit implicated key residues involved in porphyrin binding and catalysis. The work presented in this thesis has expanded our understanding of the magnesium chelatase, particularly in respect to the H subunit. With three different proteins and three substrates, it is clear that magnesium chelatase is an elaborate molecular machine that is proving to be far more complicated than ever expected.