The mechanism of molybdenum cofactor-insertion into apo-enzymes of the sulfite oxidase-class

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In the previous part of the project we had focused on the molybdenum cofactor (Moco)- binding proteins mediating transfer of Moco between the site of its synthesis and the final user enzymes. Now in the second project phase, we will focus in detail on the mechanism of Moco insertion itself. For a better understanding of Moco insertion into target enzymes it is essential to have well characterized systems available both for the Moco donor and for the Moco-free acceptor. There are a number of broad objectives that we wish to achieve: (1) Unveil the mechanism by which Moco is formed on the Moco donor protein Cnx1 (Arabidopsis) and Nit-9 (Neurospora). (2) Characterize the Moco-free form of the acceptor protein sulfite oxidase from Neurospora. In contrast to plants, Neurospora possesses the animal-like two-domain form of sulfite oxidase consisting of a Moco-domain and a hemedomain. (3) Unveil the mechanism by which Moco is inserted into apo-sulfite oxidase. Like in humans and other mammals, mature and functional Neurospora sulfite oxidase is localized in the intermembrane space of mitochondria. At what stage and where (cytosol? intermembrane space?) Moco becomes inserted? Which machinery provides Moco?