NMR study of the molecular and electronic structure of the heme cavity of Aplysia metmyoglobin. Resonance assignments based on isotope labeling and proton nuclear Overhauser effect measurements

Abstract

The 1H NMR characteristics of the high-spin metmyoglobin from the mollusc Aplysia limacina have been investigated and compared with those of the myoglobin (Mb) from sperm whale. Aplysia metMb exhibits a normal acid----alkaline transition with pK approximately 7.8. In the acidic form, the heme methyl and meso proton resonances have been assigned by 1H NMR using samples reconstituted with selectively deuterated hemins and in the latter case by 2H NMR as well. On the basis of the methyl peak intensities and shift pattern, heme rotational disorder could be established in Aplysia Mb; approximately 20% of the protein exhibits a reversed heme orientation compared to that found in single crystals. Three meso proton resonances have been detected in the upfield region between -16 and -35 ppm, showing that the chemical shift of such protons can serve as a diagnostic probe for a pentacoordinated active site in hemoproteins, as previously shown to be the case in model compounds. The temperature dependence of the chemical shift of the meso proton signals deviates strongly from the T-1 Curie behavior, reflecting the presence of a thermally accessible Kramers doublet with significant S = 3/2 character. Nuclear Overhauser effect, NOE, measurements on Aplysia metMb have provided the assignment of individual heme alpha-propionate resonances and were used to infer spatial proximity among heme side chains. The hyperfine shift values for assigned resonances, the NOE connectivities, and the NOE magnitudes were combined to reach a qualitative picture of the rotational mobility and the orientation of the vinyl and propionate side chains of Aplysia metMb relative to sperm whale MbH2O.(ABSTRACT TRUNCATED AT 250 WORDS)

Deciphering the Structural Role of Histidine 83 for Heme Binding in Hemophore HasA

“…S2B, hyperfine-shifted resonances exhibit a Curie behavior, indicative of a pure HS state (39). The meso 1 H shifts are characteristically downfield (ϳ40 ppm) for the hexacoordinate HS ferric complexes and upfield (Ϫ20 to Ϫ70 ppm) for the pentacoordinate high spin ferric complexes (39,47). The 1 H NMR spectrum of holoH83A clearly locates meso H-peak at ϳϪ25 ppm.…”

Kinetic characterization of the acid—alkaline transition in Dolabella auricularia ferric myoglobin using 1H‐NMR saturation transfer experiments

“…Peaks indicated by * and those at 25--40 ppm exhibit less than a single proton intensity and the latter disappear at acidic pH. The spectral pattern of Dolabella metMb at acidic pH is quite similar to that of the acidic form of Aplysia metMb [9]. Therefore the heme methyl proton resonances of Dolabella metMb may be assigned as A(8-CH3), B(5-CH~), C(3-CH3), and D(1-CH3) and then peaks labeled by * are attributed to the reversely oriented heine arising from the heme orientational disorder [14], based on the signal assignments tbr Aplysia metMb 8.67.…”

“…At pH 7.14, heine methyl proton signals, peaks A-D, and single proton signals, peaks a-g, are observed. The linewidth (-200 Hz) of the heme methyl proton resonance of Dolabella metMb is much smaller than that of wellcharacterized sperm whale metMb and is comparable to that of Aplysia metMb [9]. Peaks indicated by * and those at 25--40 ppm exhibit less than a single proton intensity and the latter disappear at acidic pH.…”

“…It has been shown that the heme meso-proton resonance can be used as a marker signal for identifying the coordination number of the hem¢ iron in mgtMb [9,16]. The resemblance in the spectral features between Aplysia and Dolabella metMbs, together with the observation of the marker heine meso-proton signal at -,-25 ppm (results not shown) and the slower acid-alkaline transition rate, strongly suggests the absence of the Fe-bound water molecule in Dolab¢lla metMb.…”

“…The rapid acid-alkaline transition rate for ferric myoglobin (metMb) of horse [4] and sperm whale [5] has been interpreted in terms of the ionization process of the distal His imidazole. On the other hand~ the molecular mechanism of the relatively slow rate in the sea hare, Aplysia ltmacina [8,9], or the shark, Galeorhinus japoni. cus [10], Mbs which possess the distal Val and Gin residues, respectively, are not fully understood at pres.…”