Non-Kramers ESEEM of integer-spin diferrous carboxylate-bridged clusters in proteins

The diferrous oxidation level of carboxylate-bridged non-heme diiron proteins is pivotal because only it reacts directly with dioxygen. In this state each ferrous ion has S(T) = 2. Such integer-spin, so-called 'Non-Kramers' (NK) systems typically are difficult to access by paramagnetic resonance techniques, and this property has precluded the application of ENDOR and ESEEM spectroscopies to this state. Integer-spin systems frequently exhibit a ground state 'Non-Kramers doublet', however, which is split in zero applied field by an energy within the microwave range, thus rendering the center EPR active. Recently, we showed that ENDOR and ESEEM spectroscopies also can be applied to NK doublets, in preliminary measurements on diferrous methane monoxygenase hydroxylases (MMOH(red)) Methylococcus capsulatus (Bath) and Methylosinus trichosporium OB3b and azido-hemerythrin (N₃-Hr(red)) (Hoffman, B.M.; Sturgeon, B.E.; Doan, P.E.; DeRose, V.J.; Liu, K.E.; Lippard, S.J., J. Am. Chem. Soc. 1994, 116, 6023-6024). Building on this study plus subsequent theoretical work (Hoffman, B.M., J. Phys. Chem. 1994, 98, 11657-11665), we now present a comparative 3-pulse ESEEM study of the diiron(II) centers of the two MMOH(red), N₃Hr(red), N₃Ribonucleotide reductase (N₃R2(red)), N₃Rubrerythrin (N₃Rr(red)), and N₃-'Chopped' Rubrerythrin (N₃CRr(red)), which lacks the 39-residue C-terminal FeS₄ domain. This report first presents the theory necessary for quantitative interpretations of the frequency-domain (ENDOR-like) spectra obtained by three-pulsed ESEEM measurements performed in the limit where the external magnetic field (B(o)) oriented parallel to the microwave field is small: B(o) ≤ 20 G. It then presents experimental NK-ESEEM results for ¹⁴N nuclei from coordinated histidine of each of the five diferrous proteins and from azide of the three azido-proteins. The measurements yield ¹⁴N quadrupole tensors to high precision as well as estimates of hyperfine couplings. In addition they provide otherwise unavailable information about the orientations of the tetragonal crystal fields of the individual ferrous ions. The data suggest that the histidyl ligands to the cluster play a major role in determining the electronic structure of the functionally important diferrous state of these proteins. They disclose a surprising reorientation of azide in the Rr(red) pocket upon deletion of the C-terminal domain.