ENDOR Subgroup

14/15N ENDOR spectroscopy reveals that the radical SAM enzyme MoaA positions the substrate for reaction by directly binding a purine ring nitrogen to one of the [4Fe-4S] clusters. By comparing ENDOR data from the native substrate and an alternative substrate, one differing only by a missing exocyclic amino group, the nitrogen binding site was determined. The study also suggests that binding to the cluster induces tautomerization of the substrate to the less favored form, and implies an intriguing mechanistic role for such behavior. (Collaboration with Mike Johnson (Georgia))

We study the electronic, magnetic, and structural properties of metalloenzymes and model complexes through use of advanced paramagnetic resonance techniques

 

The Hoffman Group

Application of EPR and ENDOR spectroscopy to cryoreduced (reduction by irradiation of a frozen solution with γ-rays) samples of gsNOS reveals that compound I (6) is the active heme species responsible for stage I reduction of substrate L-arginine to NOHA, but in stage II, reduction of NOHA to citruline and HNO/NO-, the reactive species is the hydroperoxo-ferriheme, 5. (Collaboration with Brian Crane (Cornell))

A combined EPR and computational study of the species corresponding to the first and last steps of the only known inorganic complex capable of catalytically reducing dinitrogen leads to a comprehensive description of their magnetic and electronic properties. In turn, this knowledge has contributed to our understanding of how trigonal coordination at Mo contributes to activation of the metal for N2 binding. (Collaboration with Richard Schrock (MIT) and Frank Neese (Bonn))

Molybdenum-dependent nitrogenase binds and reduces N2 and alkynes at the [Fe7, Mo, S9, X, homocitrate] iron-molybdenum cofactor (FeMo-co). Constraints derived from ENDOR studies of biomimetic complexes with known structure powerfully contribute to our ability to use experimentally derived ENDOR parameters for nitrogenase intermediates to reveal the structures of  the N2-derived species bound to FeMo-co during NH3 formation. The first report of a paramagnetic Fe-S compound that could bind reduced forms of N2 involved Fe complexes stabilized by the bulky β-diketiminate ligand (Vela, J.; Stoian, S.; Flaschenriem, C. J.; Münck, E.; Holland, P. L. J. Am. Chem. Soc. 2004, 126, 4522-4523). The mixed-valence FeIIFeIII complex contains a bridging phenylhydrazido (PhNNH2) ligand, a structure that of a corresponding nitrogenase intermediate. This complex was characterized by ENDOR spectroscopy using both 15N and 2H labeled as well as natural-abundance forms of the hydrazido ligand. In concert with semiempirical and DFT computations, this study provided a full set of hyperfine and quadrupole parameters for the -N-NH2 moiety. The results support the use of DFT computations in identifying nitrogenous species bound to FeMo-co of nitrogenase turnover intermediates and demonstrate that 14N quadrupole parameters from nitrogenase intermediates can provide a strong indication of the nature of the bound nitrogenous species. (Collaboration with Patrick Holland (U. of Rochester))

Useful resources about EPR and ENDOR

1.  Abragam, A, Bleaney, B. Electron Paramagnetic Resonance of Transition Ions. Oxford University Press, Oxford, 1970.

2.  Hoffman, B.M., Electron-nuclear double resonance spectroscopy  (and electron spin-echo envelope modulation) in bioinorganic chemistry, Proc. Natl. Acad. Sci., 2003, 100, 3575-3578.

3.  Lowe, D.J., ENDOR and EPR of Metalloproteins, R.G. Landes, Austin, TX.

4.  Schweiger, A., Jeschke, G., Principles of pulse electron paramagnetic resonance, Oxford University Press, Oxford, U.K., 2001.

Funding

Metalloenzymes

Enzyme Model Complexes

Hoffman Group