Solution 1H NMR investigation of the seating and rotational "hopping" of centrosymmetric etioheme-I in myoglobin: effect of globin origin and its oxidation/spin state on heme dynamics

Abstract

Solution 1H NMR spectroscopy was used to investigate the heme active-site structure and dynamics of rotation about the Fe-His bond of centrosymmetric etioheme-I reconstituted into sperm whale and horse myoglobin (Mb). Comparison of the NOESY cross-peak pattern and paramagnetic relaxation properties of the cyanomet complexes confirm a heme pocket that is essentially the same as Mb with either native protoheme or etioheme-I. Dipolar contacts between etioheme and the conserved heme pocket residues establish a unique seating of etioheme that conserves the orientation of the N-Fe-N vector relative to the axial His plane, with ethyl groups occupying the vinyl positions of protoheme. Saturation transfer between methyls on adjacent pyrroles in etioheme-reconstituted horse Mb in all accessible oxidation/spin states reveals rotational hopping rates that decrease dramatically with either loss of ligands or reduction of the heme, and correlate qualitatively with expectations based on the Fe-His bond strength and the rate of heme dissociation from Mb. The rate of hopping for etioheme in metMbCN, in contrast to hemes with propionates, is the same in the sperm whale and horse proteins.

Reactions of HNO with Heme Proteins: New Routes to HNO−Heme Complexes and Insight into Physiological Effects

“…However, if an HNO-Fe II Mb sample prepared by this method is air oxidized to Fe III Mb, and the HNO-adduct is then regenerated using the trapping methodology, only single resonances are observed in the two characteristic regions. A similar doubling of resonances was previously perceived for reconstituted metmyoglobin adducts and was attributed to heme orientational disorder;22-24 the vinylic side groups of the heme are asymmetrical, and thus flipping of the heme within a protein pocket will produce two different diastereomers. To examine this hypothesis, a sample of apomyoglobin was reconstituted with the iron complex of symmetrical 2,4-dimethyl deuteroporphyrin (Scheme 3), and its HNO adduct was prepared by the nitrite/borohydride method.…”

1H and 13C NMR Investigation of the Influence of Nonligated Residue Contacts on the Heme Electronic Structure in Cyanometmyoglobin Complexes Reconstituted with Centro- and Pseudocentrosymmetric Hemins

“…For the other hemins, there is no unique numbering system intrinsic to the hemin so that the methyls are labeled solely by the positions a-h they occupy in the protein matrix, as determined and discussed in detail previously. 40,41,43 Hence, resonances in Figure 2 are labeled M i (methyl, H i (single proton)), where i is the position on the heme periphery (M 1 for the hemin-based position or M a for the protein-based position, methyl at position 1 or position a) or the residue (i.e., H CD1 is C ζ H of Phe CD1, and M FG5 , H FG5 are methyl and proton of Ile FG5). The key dipolar shifted heme pocket residues needed 16,28,[30][31][32] to determine the magnetic axes and define heme orientation 30 are Phe43(CD1), His64(E7), Val68(E11), Ala71(E14), Leu89(F4), His93(F8), His97(FG3), and Ile99(FG5).…”

“…43 The detailed orientation of a modified hemin about its normal relative to the protein matrix can be determined from the relative magnitudes of the steady-state NOEs from methyls at position a(M a ) and h(M h ) relative to the E helix backbone Val68(E11) C R H and Ala71(E14) C β H 3 , or from methyls at positions c(M c ) and e(M e ) relative to Ile99(FG5) C γ H 3 and C δ H 3 , respectively. 28,30,43 These NOE patterns have been shown 40,41 to be essentially unchanged for hemins 2, 6, 7, and 8 relative to WT (hemin 1). Steady-state NOEs upon saturating these methyls in metMbCN reconstituted with hemin 3-5 reveal changes in relative NOEs of < 20% which translate to changes in orientation of < 3°about the heme normal 30,40,41 (not shown).…”

“…32 The heme and numerous heme pocket residue proton signals for metMbCN reconstituted with hemins 1-8 have been assigned, and the orientations of each of the hemins in the heme pocket have been reported. 32,[39][40][41][42][43]…”

Dynamic Motion and Rearranged Molecular Shape of Heme in Myoglobin: Structural and Functional Consequences

“…These NMR results suggest that heme 9 is essentially static on the NMR time scale while heme 8 is rapidly rotating. Tran and coworkers noted a possibility that the heme 9 rotates about the iron-histidine bond with a rate of 0.9–27 s −1 depending on the oxidation/spin state of the iron [25]. Since the rotation of heme 9 is slow enough on the NMR time scale, the static picture of heme 9 is a suitable approximation.…”