Proton Electron Nuclear Double Resonance from Nitrosyl Horse Heart Myoglobin: The Role of His-E7 and Val-E11

Abstract

Electron nuclear double resonance (ENDOR) spectroscopy has been used to study protons in nitrosyl horse heart myoglobin (MbNO). (1)H ENDOR spectra were recorded for different settings of the magnetic field. Detailed analysis of the ENDOR powder spectra, using computer simulation, based on the "orientation-selection" principle, leads to the identification of the available protons in the heme pocket. We observe hyperfine interactions of the N(HisF8)-Fe(2+)-N(NO) complex with five protons in axial and with eight protons in the rhombic symmetry along different orientations, including those of the principal axes of the g-tensor. Protons from His-E7 and Val-E11 residues are identified in the two symmetries, rhombic and axial, exhibited by MbNO. Our results indicate that both residues are present inside the heme pocket and help to stabilize one particular conformation.

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.…”

“…The EPR parameters for type I are given in Table II, the axial g tensor of type II was for all cases taken as g Ќ Ϸ 2.035, g ʈ Ϸ 1.98. The parameters are typical for a six-coordinated Fe 2ϩ (heme)NO structure (22)(23)(24)(25)(26)(27)(28)(29)(30)(31). The EPR spectra of the Ngb proteins could be simulated using a ratio of type I over type II (n I /n II ) equal to 80/20%, whereby this ratio was 95/5% for E7-Leu Mb.…”

“…The nature of type II is still controversial, despite the large amount of spectral data available. Several authors have ascribed type II at low temperatures to a species where the Fe-N(NO) bond aligns with the normal of the porphyrin ligand (28,26,29,39), although a topological isomer with the Fe atom displaced "below" the porphyrin ligand toward HisF8 has also been proposed (22). Recent density functional computations indicate that the observed axial g tensor agrees either with a bent end-on orientation of NO where the NO ligand eclipses one of the equatorial Fe-N porph bonds or with a partially dissociated hexacoordinated complex (distance (Fe-N(Im) Ͼ0.25 nm) with a freely rotating NO ligand (40).…”

“…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.…”

Probing the heme-pocket structure of the paramagnetic forms of cytoglobin and a distal histidine mutant using electron paramagnetic resonance

“…ENDOR studies on the A and R isomers of horse heart Mb showed that in the two forms, the NO ligand is stabilized by E7His and E11Val, but that the strength of the interaction is different [41].…”