PROBING THE STABILIZING ROLE OF C-TERMINAL RESIDUES IN TRIMETHYLAMINEDEHYDROGENASE

In trimethylamine dehydrogenase, a homodimeric iron-sulfur flavoprotei n, the C-terminal 17 residues of each subunit (residues 713-729) embra ce residues on the other subunit, The role of this unusual mode of int eraction at the subunit interface was probed by isolating three mutant forms of trimethylamine dehydrogenase in which the C-terminus of the enzyme was deleted by five residues [Delta(725-729], 10 residues [Delt a(720-729)] and 17 residues [Delta(713-729)]. The solution properties and conformational states of the three mutant enzymes were investigate d using optical, fluorescence and circular dichroism spectroscopies, A NS binding and a novel and conformationally sensitive hydrodynamic met hod. The data reveal that sequential deletion of the C-terminus of tri methylamine dehydrogenase does not affect significantly dimer stabilit y or the overall structural integrity of the enzyme. However, deletion of the C-terminus severely compromises, but does not abolish, the abi lity of the enzyme to become covalently coupled with the redox cofacto r FMN in the active site, located over 20 Angstrom from the C-terminus , Hydrodynamic studies reveal minor conformational changes in the dele tion mutants that lead to a more compact enzyme structure. These confo rmational changes are probably transmitted to the active site via alte ring the interaction of the C-terminus with the second helix in the be ta/alpha barrel of trimethylamine dehydrogenase, leading to poor flavi nylation during the folding of the enzyme and assembly with FMN.