Structure of the Dithionite-Generated Tryptophan Tryptophylquinone Cofactor Radical in Methylamine Dehydrogenase Revealed by ENDOR and ESEEM Spectroscopies

The structure of the tryptophan tryptophyl-semiquinone cofactor in frozen solutions of randomly-oriented methylamine dehydrogenase from Paracoccus denitrificans has been characterized by continuous wave-electron nuclear double resonance (CW-ENDOR) and electron spin echo envelope modulation (ESEEM) spectroscopic analyses of hyperfine and nuclear quadrupole interactions. The radical was generated by direct reduction of the oxidized enzyme with dithionite in ¹H₂O or ²H₂O buffer. Multifrequency ESEEM studies reveal weak hyperfine interactions with two nitrogen nuclei that have ¹⁴N nuclear quadrupole coupling parameters consistent with indole nitrogen present in the two tryptophan side chains that are cross-linked covalently to form the cofactor. Analysis of nonsolvent exchangeable hyperfine couplings in ¹H ENDOR powder spectra yields the rhombic hyperfine tensors for two α-¹H and three β-¹H interactions, which are assigned to the two C–H_αinteractions on the ortho-quinone-modified indole ring and the two pairs of β-methylene-¹H of the indole and indole-quinone rings. The pairs of methylene C_β–¹H bonds are oriented relative to the ring plane normals at dihedral angles, θ, of (55°, 65°) and (~60°, ~60°). Analysis of orientation-restricted ENDOR spectra shows that the indole ring planes are rotated by the dihedral angle, of χ, 48° ±11° along their covalent link. Weak matrix ²H ESEEM suggests that the unpaired spin density (ϱ_π) Is localized predominantly on the buried indole-quinone ring, away from the unmodified indole that extends to the protein solvent accessible surface. Absence of strong exchangeable a-hydrogen hyperfine couplings in ¹H ENDOR and ²H ESEEM spectra suggests that the quinonoid oxygen atoms are deprotonated and, therefore, that the radical is negatively charged. The θ and χ values in the TTQ semiquinone are the same within error as those determined previously for oxidized TTQ by X-ray crystallographic analyses. Thus, redox state changes do not trigger significant conformational changes in the cofactor or surrounding protein medium in isolated methylamine dehydrogenase. Although 87% of the detected ϱ_π is localized on the indole-quinone ring, significant ϱπIs distributed to the fused pyrrole ring of the unmodified indole, despite the suboptimal inter-ring π–π conjugation geometry. Therefore, in the native cofactor oxidation reactions, chemical transformations at the active center can influence directly the inter-ring partitioning of ϱ_π.