1H-NMR heme resonance assignments by selective deuterations in low-spin complexes of ferric hemoglobin A

doi:10.1016/0167-4838(88)90108-2

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology

Volume 952, 1988, Pages 131-141

Biochimica et Biophy...

https://doi.org/10.1016/0167-4838(88)90108-2 Get rights and content

Abstract

The heme methyl and vinyl α-proton signals have been assigned in low-spin ferric cyanide and azide ligated derivatives of the intact tetramer of hemoglobin A, as well as the isolated chains, by reconstituting the proteins with selectively deuterated hemins. For the hemoglobin cyanide tetramer, assignment to individual subunits was effected by forming hybrid hemoglobins possessing isotope-labeled hemins in only one type of subunit. The heme methyl contact shift pattern has 1-methyl and 5-methyl shifts furthest downfield in both chains and the individual subunits of the intact hemoglobin in both the cyanide- and azide-ligated species, which is consistent with a dominant rhombic perturbation due to the proximal His-F8 imidazole π bonding in the known structure for human adult hemoglobin. The individual chain and subunit assignments confirm that the detailed electronic/magnetic properties of the heme pocket are essentially unaltered upon assembling the R-state tetramer from the isolated subunits.

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Substitution of the heme binding module in hemoglobin α- and β- subunits. Implication for different regulation mechanisms of the heme proximal structure between hemoglobin and myoglobin
2000, Journal of Biological Chemistry
Citation Excerpt :
Although the cyanomet form is not biologically active in globin proteins, the1H NMR spectra afford various structural information for liganded hemoglobin (45-48). As illustrated in Fig.7, 5-, 1-methyl, and 2-vinyl Cα proton signals have been assigned for the human hemoglobin α- and β-subunits and tetrameric Hb A (49). Noteworthy here is that the spectral feature of the cyanomet-α2[βα(HBM)2] tetramer was also virtually identical to that of Hb A, indicating that the heme electronic state of the α2[βα(HBM)2] tetramer was not significantly affected by the module substitution.
In our previous work, we demonstrated that the replacement of the “heme binding module,” a segment from F1 to G5 site, in myoglobin with that of hemoglobin α-subunit converted the heme proximal structure of myoglobin into the α-subunit type (Inaba, K., Ishimori, K. and Morishima, I. (1998) J. Mol. Biol.283, 311–327). To further examine the structural regulation by the heme binding module in hemoglobin, we synthesized the βα(HBM)-subunit, in which the heme binding module (HBM) of hemoglobin β-subunit was replaced by that of hemoglobin α-subunit. Based on the gel chromatography, the βα(HBM)-subunit was preferentially associated with the α-subunit to form a heterotetramer, α₂[βα(HBM)₂], just as is native β-subunit. Deoxy-α₂[βα(HBM)₂] tetramer exhibited the hyperfine-shifted NMR resonance from the proximal histidyl N_δH proton and the resonance Raman band from the Fe-His vibrational mode at the same positions as native hemoglobin. Also, NMR spectra of carbonmonoxy and cyanomet α₂[βα(HBM)₂] tetramer were quite similar to those of native hemoglobin. Consequently, the heme environmental structure of the βα(HBM)-subunit in tetrameric α₂[βα(HBM)₂] was similar to that of the β-subunit in native tetrameric Hb A, and the structural conversion by the module substitution was not clear in the hemoglobin subunits. The contrastive structural effects of the module substitution on myoglobin and hemoglobin subunits strongly suggest different regulation mechanisms of the heme proximal structure between these two globins. Whereas the heme proximal structure of monomeric myoglobin is simply determined by the amino acid sequence of the heme binding module, that of tetrameric hemoglobin appears to be closely coupled to the subunit interactions.
Structural and functional roles of heme binding module in globin proteins: Identification of the segment regulating the heme binding structure
1998, Journal of Molecular Biology
To investigate structural and functional significance of a newly proposed structural unit in globins, the “heme binding module”, we synthesized a “heme binding module”-substituted chimeric globin and characterized its function and structure. In our previous study we proposed that the heme binding module, corresponding to the segment from Leu(F1) to Phe(G5) in hemoglobin α-subunit, plays a key role in constructing the heme proximal structure in globins. The replacement of the heme binding module in myoglobin with that of hemoglobin α-subunit converted the absorption spectra into that of the α-subunit, and, in the resonance Raman spectra, the vibration mode characteristic of myoglobin completely disappeared after the module replacement. The hyperfine-shifted NMR resonances for the cyanide-bound form of the module-substituted myoglobin also revealed that the orientation of the axial histidine is close to that of the α-subunit rather than that of myoglobin, while the deviations of the resonance positions of the NMR signals from the amino acid residues located in the distal site were subtle, supporting the preferential structural alterations in the heme proximal site. The present finding for the structural alterations in the module-substituted myoglobin confirms that the heme binding module can be a segment regulating the heme proximal structure in globin proteins.
Nmr Study Of Active Sites In Paramagnetic Haemoproteins
1998, Annual Reports on NMR Spectroscopy
NMR studies of paramagnetic haemoproteins are reviewed with special emphasis on characterization of both structural and dynamic properties of haem active site. In the past decade, the development of NMR methodologies for detecting the connections between hyperfine shifted signals has contributed greatly to establishing systematic and reliable strategies for the signal assignments of paramagnetic haemoproteins. The use of reconstituted haemoprotein is a characteristic of the study of b-type haemoproteins and NMR studies on reconstituted myoglobins and haemoglobins are described in some detail. Additionally, nonequivalence in haem electronic structure between the two different subunits in human adult haemoglobin enables one to characterize the individual subunits in intact tetramers using NMR, and the advantages in studying tetrameric haemoglobin by N M R are also described.
Time-resolved fluorescence of hemoglobin species
1997, Biophysical Chemistry
We used time-resolved fluorescence in the pico- to nanosecond time range to monitor the presence of tetramers, dimers and monomers in carbonmonoxyhemoglobin (COHb) solutions and to investigate how their distributions change under different experimental conditions. Comparison of fluorescence lifetime computed from the atomic coordinates of COHb (Vasquez et al., 1996) with those experimentally measured allowed identification of molecular species present in the hemoglobin solution. It was possible to observe modification of the distribution of tetramers, dimers, monomers and species with disordered hemes produced by different experimental conditions. Protein concentration affected the detectable lifetimes, indicating increasing amounts of dimers and monomers at low protein concentrations, while the amount of inverted hemes was not modified. Titration with up to 1 M NaCl modified only the extent of dissociation of hemoglobin into dimers, without affecting heme inversion and monomer formation. Hyperbaric pressure increased the amounts of dimers and monomers. This is the first time that monomeric subunits of hemoglobin have been detected at neutral pH in the normal system.
<sup>1</sup>H nuclear magnetic resonance study of the prosthetic group in sulfhemoglobin
1992, Archives of Biochemistry and Biophysics
The molecular and electronic structure of the modified prosthetic group of sulfhemoglobin (SHb) was investigated by ¹H NMR for the low-spin ferric cyano-met and high-spin ferrous deoxy sulfhemoglobin complex. The ¹H NMR resonances of the two subunits in the cyano-met SHb complex were differentiated on the basis of the differential stability toward regeneration of native subunits. The subunit origin for the two sets of resonances was established by formation of the sulfglobin protein for the isolated α-chain prior to assembling with the native β-subunit to yield a tetramer with sulfhemin in the α-subunits. The subunit peak assignments establish that it is the β-subunit of SHb which regenerates more rapidly to native protein. The hyperfine shifted sulfhemin peaks were assigned based on steady-state nuclear Overhauser effects which demonstrated that similarly hyperfine shifted peaks exhibit the same dipolar connectivities observed in the analogous sulfmyoglobin complex. Hence it is concluded that pyrrole B is the site of reaction in both hemoglobin and myoglobin. The initially formed SHb complex failed to equilibrate to yield a complex with a sulfhemin sufficiently stable to extraction as found previously for sulfmyoglobin. However, apoHb readily bound the green sulfhemin extracted from the terminal alkaline equilibration product of sulfmyoglobin. The inhibition on the equilibration to the alkaline form with the exocyclic thiolene ring is attributed to the interaction with Val FG5. The observations of the same dipolar connectivities among similarly hyperfine shifted peaks in the directly prepared and reconstituted SHb complexes further support the same structure for the sulfhemin in sulfmyoglobin and SHb. The strongly hyperfine shifted peaks in the deoxy form of both SHb complexes were found very similar to those of the analogous sulfmyoglobin complexes. The proximal His labile ring proton signal appears to experience a 5- to 10-ppm decrease upon conversion of a native globin to sulfglobin. This attenuation may provide a probe for differentiating chlorins and hemins in globin pockets.
<sup>1</sup>H NMR resonance assignments in a paramagnetic heme protein by two-dimensional spectroscopy: Heme resonances in equine met-azido myoglobin
1991, Biochemical and Biophysical Research Communications
Specific heme protons for the majority of resonances in the downfield resolved region of equine met-azido myoglobin have been assigned using solely the two-dimensional ¹H NMR experiments NOESY and COSY. Met-azido myoglobin provides a useful test case for the the applicability of these techniques to paramagnetic proteins for the following reasons. First met-azido myoglobin is a mixed spin-state protein, with significantly shorter relaxation times and broadened lines relative to pure low-spin systems (eg., met-cyano myoglobin). Second, met-azido hemoglobin and met-azido myoglobin are important as models for the physiological forms of hemoglobin. Third, a few sperm whale met-azido myoglobin resonances have been previously assigned, which permits a comparison of assignments for these similar proteins, and a check of the method presented here.

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1H-NMR heme resonance assignments by selective deuterations in low-spin complexes of ferric hemoglobin A

Abstract

Methods Enzymol.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

J. Mol. Biol.

J. Mol. Biol.

J. Biol. Chem.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

J. Mol. Biol.

Biochim. Biophys. Acta

J. Biol. Chem.

J. Mol. Biol.

J. Biol. Chem.

J. Mol. Biol.

Hemoglobins and Myoglobins in Their Reactions With Ligands

Struct. Bonding

CRC Crit. Rev. Biochem.

Annu. Rep. NMR Spectra

J. Mol. Biol.

¹H-NMR heme resonance assignments by selective deuterations in low-spin complexes of ferric hemoglobin A