TTQ cofactor

The identification of a thio-ether bond in the active site of GOase (Ito et al., 1991) immediately encouraged speculation about its biogenesis. Is the formation of this bond autocatalytic as appears to be the case with the TPQ cofactor in amine oxidases (Dooley, 1999) or does it require chaperones as is the case in the biogenesis of the TTQ cofactor (Christoserdov et al., 1991) Does biogenesis of the thioether bond involve radicals There are... 

Three-dimensional structures. The TPQ-con-taining amine oxidase from E. coU is a dimer of 727-residue subunits with one molecule of TPQ at position 402 in each subunit. 7458 Methylamine dehydrogenase is also a large dimeric protein of two large 46.7-kDa subunits and two small 15.5-kDa subunits. Each large subunit contains a TTQ cofactor Reduced TTQ is reoxidized by the 12.5-kDa blue copper protein amicyanin. Crystal structures have been determined for complexes of methylamine dehydrogenase with amicyanin and of these two proteins with a third protein, a small bacterial cytochrome... 

Figure 5 Structure of the TTQ cofactor in methylamine dehydrogenase from P. denitrificans. In this enzyme oxygenation of /3Trp57 and cross-linking with /3Trp108 yields the TTQ cofactor, which is displayed as sticks colored gray for carbon, red for oxygen, and blue for nitrogen. The coordinates from PDB entry 2bbk were used to display this structure.

The reductive half-reaction of methylamine dehydrogenase is shown in Scheme 10. The methylamine substrate initiates a nucleophilic attack on the quinone carbon at the C6 position of the TTQ cofactor displacing the oxygen to form a substrate-TTQ Schiff base adduct (29). The reactivity of the C6 position was demonstrated by covalent adduct formation at this position by hydrazines which are inactivators of methylamine dehydrogenase. Deprotonation of the substrate-derived carbon of 29 by an active-site amino acid residue results in reduction of the cofactor and yields an intermediate in which the Schiff base is now between the nitrogen and substrate-derived carbon (30). Hydrolysis of 30 releases the formaldehyde product and yields the aminoquinol form of the cofactor with the substrate-derived amino group still covalently bound (31). 

Fig. 14. Structure of the tryptophan-derived TTQ cofactor of MADH. The covalent attachment to the protein through the O atoms of the modified tryptophans is also shown.

Fig. 16. Stereoview of the TTQ cofactor and surrounding residues. In bold, Trp-108, Trp-59, and Asp-74 disulfide bridges are shown between Cys-109 and Cys-78 (left) and between Cys-77 and Cys-121 (right).

Before reviewing the details of the reactions between proteins from P. denitrificans it is important to first discuss the available structural information. The proteins MADH and amicyanin are known to associate quite strongly in solution (288). A binary complex has been crystallized and the structure (92) of part of the interface between the two proteins is shown in Fig. 20. The hydrophobic patch of amicyanin, which surrounds the exposed imidazole ring of the ligand His-96, is found associating with a mainly hydrophobic region on the L subunit of MADH. There are also interactions between amicyanin and the larger H subunit of MADH. The two proteins interact in such a way that the TTQ cofactor of MADH and the copper of amicyanin are approximately 9 A apart. 

Fig. 20. Position of the TTQ cofactor of MADH and the copper of amicyanin in the crystal structure of the binary complex of the two proteins. The distance from the edge of the TTQ cofactor to the Cu atom is 9.3 A. The side chain of His-96 is positioned between the Cu and the TTQ. The interface between the two proteins in the complex is formed by the hydrophobic patch of amicyanin and a similar hydrophobic surface of MADH, surrounding the exposed edge of the TTQ cofactor.

We start our study by investigating a model of the biological TTQ cofactor in the gas phase (Figure 5.10). We consider two redox states, the fully reduced,... 

Figure 5.10 Model of the TTQ cofactor. TTie average distances of the bonds whose length vary by more than 0.01 A between the two redox states are shown. These distances were obtained from around 25 ps of gas phase DFT-BOMD simulations respectively of the anionic and radical forms (first and second lines).

Figure 5.11 Decay of the overlap contribution to decoherence within the TTQ cofactor in gas phase. Left total overlap function (full line). Right definition of contributions of the three atom groups (dashed/dotted lines). The circled line is the product of the different contributions. All the curves are real parts of the function averaged over the sets of diverging DFT-BOMD trajectories.

Having identified the intramolecular mechanism of decoherence within the TTQ cofactor in gas phase, we now investigate the influence of its environment. Within the MADH enzyme, the TTQ cofactor is surrounded on one hand by protein residues and on the other hand by water molecules. To include these... 

Figure 5.12 Average decay of the overlap term for the TTQ cofactor time in the gas phase and in water. SPCF, flexible simple point charge.

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