Binding of N-15-labeled isoniazid to KatG and KatG(S315T): Use of two-spin[zz]-order relaxation rate for N-15-Fe distance determination

Isoniazid has been used to treat tuberculosis for over 40 years, but its me chanism of action is not yet fully understood. It is known that a catalase/ peroxidase KatG is required for isoniazid activation. A point mutation in K atG, KatG(S315T), is found in > 50% of isoniazid-resistant strains. In this study we have measured the distance between the N-15 labeled amide nitroge n of isoniazid and the Fe ion in the active site of KatG and KatG(S315T). T he distances are found to be equal within experimental error, 3.8 +/- 0.8 a nd 4.4 +/- 0.9 Angstrom, respectively. A new method of measuring longitudin al relaxation rates of insensitive nuclei in paramagnetic systems via zz-or der is proposed. The longitudinal relaxation rate of the N-15 nucleus was o btained from the independently measured longitudinal proton relaxation rate and the longitudinal zz-order relaxation rate of scalar coupled N and H at oms. To eliminate cross-correlations of different relaxation sources, a rem ote proton was used to create zz-order and detect the N-15 nucleus. The obt ained N-15-Fe distances are significantly shorter than previously reported H-1-Fe distances (Wengenack, N. L.; Todorovic, S.; Yu, L; Rusnak, F. Bioche mistry 1998, 37, 15825), indicating that the isoniazid molecule approaches the heme Fe ion via the hydrazine nitrogen atoms. The proposed method for t wo-spin order relaxation measurements is quite general and can be used to p robe the distance between insensitive nuclei and a paramagnetic center in v arious protein-substrate complexes.