Unambiguous Determination of theg-Matrix Orientation in a Neutral Flavin Radical by Pulsed Electron−Nuclear Double Resonance at 94 GHz

Abstract

The recent observation of photoinduced radical pairs comprising a flavin radical and an oxidized amino acid residue in various blue-light-sensitive proteins has highlighted the need to gain a more complete understanding of the electronic structure of flavin radicals. In particular, precise knowledge of the anisotropy of the Zeeman interaction quantified by the g-tensor is necessary for attaining an unambiguous identification of flavin radicals by electron paramagnetic resonance (EPR). In a recent study of a protein-bound neutral flavin radical, we have determined the principal values of the g-tensor using high-frequency/high magnetic field EPR performed at 360 GHz/12.8 T. However, in those experiments, the orientation of the principal axes of g could not be unambiguously established with respect to the molecular frame of the isoalloxazine moiety. In this contribution we resolve this ambiguity by pulsed electron-nuclear double resonance (ENDOR) at 95 GHz/3.5 T (W-band). At such high values of the microwave frequency and the magnetic field, the g anisotropy provides improved spectral resolution compared to an ENDOR experiment performed at conventional 9.5 GHz/0.35 mT (X-band). This enables one to utilize Zeeman magnetoselection to obtain single-crystal-like data from disordered samples in frozen solution. Experiments exploiting this orientation selection have allowed us to use the hyperfine coupling of the methyl protons at C(8alpha) of the isoalloxazine ring to determine the angle between the molecular frame and the principal axes of g. Quite surprisingly, the g-tensor in FADH* is not oriented as one would have expected for a 1,3-semibenzoquinone radical. For the latter, the X-axis of g commonly bisects the smaller angle between the two axes along the C=O bonds. In FADH*, the large spin density on N(5) and C(4a) apparently contributes to a significant (44 degrees ) reorientation of the g-tensor axes.