Characterization of the tryptophan tryptophyl-semiquinone catalytic intermediate of methylamine dehydrogenase by electron spin-echo envelope modulation spectroscopy

The electronic structure of the tryptophan tryptophyl-semiquinone (TTQ(.)) cofactor generated by addition of the substrate, methylamine, to methylamin e dehydrogenase (MADH) from Paracoccus denitrificans has been studied by co ntinuous-wave electron paramagnetic resonance (cw-EPR) and electron spin-ec ho envelope modulation (ESEEM) spectroscopy. The cw-EPR spectrum of TTQ sem iquinone prepared by substrate addition (N-form) was found to differ substa ntially from that observed when the semiquinone was generated by dithionite reduction of the enzyme (O-form). These differences prompted a detailed st udy of hyperfine and nuclear quadrupole interactions of the three N-14 atom s Of the semiquinone species using ESEEM. Two of these heteroatoms are deri ved from the indole and indole-quinone side chains that comprise TTQ, while the third N-14 originates from substrate methylamine. Three-pulse ESEEM sp ectra of the (CH3NH2)-N-14-reduced sample showed three isolated features at 1.0, 1.5, and 4.3 MHz, which were absent in the MADH sample reduced with ( CH3NH2)-N-15. Analysis of the spectral data for substrate-derived N-14 reve aled an isotropic hyperfine coupling of 2.4 MHz and nuclear quadrupole coup lings characterized by e(2)qQ = 1.7 MHz and eta = 0.5. The hyperfine and th e nuclear quadrupole couplings found for the two N-14 nuclei indigenous to TTQ were: A(iso) 2.8 and 1.9 MHz; e(2)qQ, 3.0 and 2.1 MHz and eta, 0.3 and 0.7, respectively. Taken together, these couplings provide definitive evide nce that substrate N-14 is covalently bound to TTQ when the cofactor is in its one-electron reduced form and that it has an imine-like structure. The intensities of the:modulations indicate that the semiquinone generated by t he method recently reported by Zhu and Davidson (Biochim. Biophys. Acta 199 8, 1364, 297-300) results in a homogeneous preparation of the radical. A co mparison of the N-14 hyperfine and nuclear quadrupole couplings measured fo r the N-form semiquinone with those measured previously for O-form (Warncke , K.; Brooks, H. B.; Lee H.-I.; McCracken, J.; Davidson, V. L.; Babcock, G. T. J. Am. Chem. Sec. 1995, 117, 10063-10075) shows that a significant chan ge occurs in the highest occupied molecular orbital when substrate nitrogen is bound, and may be related to the different redox and electron-transfer properties of these two semiquinone forms.