Crystallographic Study of a Quinoprotein Electron Transfer System: Methylamine Dehydrogenase

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Mathews, F.S.MCB-0091084Electron transfer between biological macromolecules is fundamental to many biological processes. It usually occurs within a transient complex in which the participating redox centers and the intervening protein are optimally configured to promote and regulate electron flow between the centers. Such complexes are difficult to study structurally since they are intrinsically unstable and usually cannot be crystallized. Methylamine dehydrogenase (MADH), amicyanin and cytochrome c551i from Paracoccus denitrificans form one of the best characterized physiological electron transfer complexes and is the only complex of three soluble redox proteins for which a high resolution structure is available. MADH is an .2 .2 heterodimer of 124 kDa and contains the unusual redox 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. Aromatic amine dehydrogenase (AADH) from Alcaligenes faecalis is similar to MADH in size and quaternary structure and also contains TTQ as it redox cofactor. However, its specific electron acceptor is an azurin and neither MADH nor AADH will efficiently reduce the alternative acceptor protein, azurin nor amicyanin, respectively. The aims of this proposal are (1) to refine the structures of oxidized and reduced amicyanin at both pH 5.5 and 8.0 at atomic resolution (1 .or below), (2) carry out structural studies of additional redox and pH variants and catalytic intermediates of the MADH/amicyanin and MADH/amicyanin/c551I complexes, (3) structurally characterize mutants of amicyanin and of MADH within these complexes, (4) further analyze redox and cation-bound states of MADH from Methylobacillus flagellatum KT and Methylobacterium extorquens AM1, and (5) complete the structure analysis of AADH and of the binary complex between AADH and azurin.The proposed studies of the MADH and AADH enzyme systems will help identify structural features that are important for electron transfer, including those involved in partner recognition, control of electron transfer and determining paths for electron flow within the complexes. In addition, the structural studies of the mechanism of substrate oxidation by the TTQ cofactor will help 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.