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 he moglobin with asymmetrically ligated alpha(beta)-chains, NO-myoglobin, and NO-Fe2+(TPP)-imidazole model complexes. The ESEEM spectra of the native com plexes 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 a n almost purl state, i.e., 80% "state I" and 90% "state II", respectively. These results correlate well with the two-conformation model of 6-coordinat ed NO-heme complexes proposed earlier from the evaluation of temperature-de pendent EPR/ENDOR spectra (Morse, R. H.; Chan, S. I. J. Biol. Chem. 1980, 2 55, 7876. Huttermann, J.; Burgard, C.; Kappl, R. J. Chem. Sec., Faraday Tra ns. 1994, 90, 3077). Application of two-dimensional ESEEM spectroscopy (HYS CORE) to the isolated alpha NO and beta NO chains allowed the characterizat ion of the pyrrole nitrogen HFI in both conformations. A third nitrogen cou pling was identified in the HYSCORE of the beta NO chain. It was tentativel y 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. Th ese 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 physiolog ical role of NO related to its interactions with protein metal centers.