Crystallographic Study of a Quinoprotein Electron Transfer System: Methylamine Dehydrogenase

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9419899 Mathews Structural studies will be carried out on the quinoprotein-electron transfer system methylamine dehydrogenase (MADH) from Paracoccus denitrificans by the methods of x-ray diffraction. Three proteins are involved in this system, MADH, amicyanin and cytochrome c551i. MADH is an H2L2 dimer of 124 kDa and contains the unusual cofactor tryptophan tryptophylquinone (TTQ). Amicyanin is a blue copper protein of 12.5 kDa which interacts specifically with MADH. Cytochrome c551i is a 17.5 kDa protein which can accept electrons from amicyanin and transfer them via another cytochrome to a terminal oxidase. The structures of MADH, amicyanin, the binary complex of MADH with amicyanin and the ternary complex of MADH, amicyanin and cytochrome c551i have been determined. Refinement of the present complexes will be extended to higher resolution by cryo-cooling and use of synchrotron radiation. Mutants of amicyanin will be studied both alone and in the complexes. Studies of MADH and its electron transfer complexes will also be carried out after reaction with substrates and inhibitors and in different redox states. The structure of MADH from Methylophilus W3A1, which uses a cytochrome rather than amicyanin as the primary electron acceptor, will be completed and compared with the Paracoccus enzyme. Finally, attempts will be made to co-crystallize the W3A1 enzyme with a cytochrome electron acceptor from the same organism. %%% Electron transfer is fundamental to many biological processes, but is difficult to study structurally since the components are usually insoluble and cannot be crystallized. The proposed studies of the MADH enzyme system will provide valuable information about this process. The interaction of the three components of this soluble electron transport system define molecular features important for recognition and control of electron transfer and define likely pathways for electron flow. The proposed mutational and ligand- induced perturbati ons of the system will further probe this complex precess. In addition, the molecular details of substrate oxidation by the TTQ cofactor will shed light on how this cofactor functions in vivo and how it differs from other, more common redox cofactors. TTQ is unusual because it is obtained directly from the fusion of two amino acid side chains coded by genomic DNA rather than from a separate biosynthetic pathway. The MADH system is well suited to provide an understanding of these important processes at the molecular level. ***