Characterization of Bimodal Coordination Structure in Nitrosyl Heme Complexes through Hyperfine Couplings with Pyrrole and Protein Nitrogens

Abstract

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 α(β)-chains of hemoglobin, two hybrids of hemoglobin with asymmetrically ligated α(β)-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 αNO and βNO chains were found in an almost pure 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. Hüttermann, J.; Burgard, C.; Kappl, R. J. Chem. Soc., Faraday Trans. 1994, 90, 3077). Application of two-dimensional ESEEM spectroscopy (HYSCORE) to the isolated αNO and βNO chains allowed the characterization of the pyrrole nitrogen HFI in both conformations. A third nitrogen coupling was identified in the HYSCORE of the βNO chain. It was tentatively assigned to the Nε 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 surrounding and could provide an important contribution to the discussion on the physiological role of NO related to its interactions with protein metal centers.

Nitric Oxide Binding Properties of Neuroglobin

“…The spectra were recorded directly after freezing the same amounts of the living cell cultures. The spectra are dominated by an orthorhombic EPR signal, which is typical for a six-coordinated Fe 2ϩ ⅐heme(NO) complex (22)(23)(24)(25)(26)(27)(28)(29)(30). Repeated experiments revealed that for the same quantity of cells, the EPR intensity was found to be a factor 4 -6 higher for the cell cultures overexpressing mutant Ngb than for those overexpressing wt Ngb (see Fig.…”

“…Electron paramagnetic resonance (EPR) has been used for more than thirty years to analyze nitrosyl (NO) ligation to the heme group of different hemeproteins (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). The NO radical bound to the central Fe 2ϩ ion in a hemeprotein forms a paramagnetic, low-spin (S ϭ 1 ⁄2) complex, which can be detected by EPR.…”

4-Demethylwyosine Synthase from Pyrococcus abyssi Is a Radical-S-adenosyl-l-methionine Enzyme with an Additional [4Fe-4S]+2 Cluster That Interacts with the Pyruvate Co-substrate

“…where N is the nuclear Zeeman frequency, a N is isotropic hyperfine coupling constant, K is the quadrupole coupling constant, and is the asymmetry parameter (21).…”

The [Fe-Fe]-Hydrogenase Maturation Protein HydF from Thermotoga maritima Is a GTPase with an Iron-Sulfur Cluster

“…They correspond to ⌬I ϭ 2 nuclear transitions and are, to the first order, insensitive to orientation broadening. It is possible to obtain a rough estimate of the hyperfine coupling constant a (supposed to be mainly isotropic) and quadrupolar coupling constant K from the frequency position of the dqdq correlation peaks using equation 1 ( being the asymmetry parameter) (37). Thus, a nitrogen atom ligated to a Fe-S cluster is easily detectable due to the strong coupling of the nuclear spin (I ϭ 1) with the unpaired electron.…”