Characterization of bimodal coordination structure in nitrosyl heme complexes through hyperfine couplings with pyrrole and protein nitrogens

Orientation-selected three-pulse ESEEM experiments have been performed on a series of nitrosyl hemoproteins: HbNO in its two quaternary (R/T) states, the isolated NO-ligated alpha(beta)-chains of hemoglobin, two hybrids of hemoglobin with asymmetrically ligated alpha(beta)-chains, NO-myoglobin, and NO-Fe2+(TPP)-imidazole model complexes. The ESEEM spectra of the native complexes clearly revealed the contribution from two conformational states of the NO-heme group. At 4.2 K the alpha NO and beta NO chains were found in an almost purl state, i.e., 80% "state I" and 90% "state II", respectively. These results correlate well with the two-conformation model of 6-coordinated NO-heme complexes proposed earlier from the evaluation of temperature-dependent EPR/ENDOR spectra (Morse, R. H.; Chan, S. I. J. Biol. Chem. 1980, 255, 7876. Huttermann, J.; Burgard, C.; Kappl, R. J. Chem. Sec., Faraday Trans. 1994, 90, 3077). Application of two-dimensional ESEEM spectroscopy (HYSCORE) to the isolated alpha NO and beta NO chains allowed the characterization of the pyrrole nitrogen HFI in both conformations. A third nitrogen coupling was identified in the HYSCORE of the beta NO chain. It was tentatively assigned to the N-epsilon nitrogen of distal His E7 which is suggested to form a hydrogen bond to the NO group in the axial NO-heme conformation. These findings support the proposal that the variation of binding geometry in two states of NO-heme is controlled by the heme's protein surroundings and could provide an important contribution to the discussion on the physiological role of NO related to its interactions with protein metal centers.