Abstract
The electronic structure of the tryptophan tryptophyl-semiquinone (TTQ•) cofactor generated by addition of the substrate, methylamine, to methylamine dehydrogenase (MADH) from Paracoccus denitrificans has been studied by continuous-wave electron paramagnetic resonance (cw-EPR) and electron spin−echo envelope modulation (ESEEM) spectroscopy. The cw-EPR spectrum of TTQ semiquinone prepared by substrate addition (N-form) was found to differ substantially from that observed when the semiquinone was generated by dithionite reduction of the enzyme (O-form). These differences prompted a detailed study of hyperfine and nuclear quadrupole interactions of the three 14N atoms of the semiquinone species using ESEEM. Two of these heteroatoms are derived from the indole and indole−quinone side chains that comprise TTQ, while the third 14N originates from substrate methylamine. Three-pulse ESEEM spectra of the CH314NH2-reduced sample showed three isolated features at 1.0, 1.5, and 4.3 MHz, which were absent in the MADH ...
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