Crystallographic Study of a Quinoprotein Electron Transfer System: Methylamine Dehydrogenase

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9728885 Mathews Structural studies will be carried out on the quinoprotein-electron transfer systems methylamine dehydrogenase (MADH) from Paracoccus denitrificans and aromatic amine dehydrogenase (AADH) by the methods of x-ray diffraction. Three proteins are involved in the first 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. AADH is an H2L2 heterotetramer of 114 kDa and also contains TTQ. Its electron acceptor is azurin, a blue copper protein of 15 kDa. Structures of the native complexes will be refined at high resolution and those of several redox variants of the binary and ternary complexes will be analyzed. Mutants of amicyanin and of MADH in the complexes will be studied to analyze the structural changes caused by these mutations. 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 refinement of two native and of two inhibited forms of MADH from Methylophilus W3A1, which uses a cytochrome rather than amicyanin as the primary electron acceptor, will be completed. A series of studies of MADH from two other organisms, Methylobacterium extorquens AM1 and Methylobacillus flagellatum KT will also be studied. Finally, the structure of AADH and its putative complex with azurin will be determined and compared with the Paracoccus enzyme. Electron transfer is fundamental to many biological processes, but is difficult to study structurally since the components are usually insoluble and cannot b e crystallized. The proposed studies of the MADH and AADH enzyme systems will provide valuable information about this process. The interaction of the various 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 perturbations of the MADH system will further probe this complex process. 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 and AADH systems are well suited to provide an understanding of these important processes at the molecular level.