Proton nuclear magnetic resonance study of the molecular and electronic structure of the heme cavity in Aplysia cyanometmyoglobin

Abstract

The 1H NMR spectrum of the low-spin, cyanide-ligated ferric complex of the myoglobin from the mollusc Aplysia limacina has been investigated. All of the resolved resonances from both the hemin and the proximal histidine have been assigned by a combination of isotope labeling, spin decoupling, analysis of differential paramagnetic relaxation, and nuclear Overhauser (NOE) experiments. The pattern of the heme contact shifts is unprecedented for low-spin ferric hemoproteins in exhibiting minimal rhombic asymmetry. This low in-plane asymmetry is correlated with the X-ray-determined orientation of the proximal histidyl imidazole plane relative to the heme and provides an important test case for the interpretation of hyperfine shifts of low-spin ferric hemoproteins. The bonding of the proximal histidine is shown to be similar to that in sperm whale myoglobin and is largely unperturbed by conformational transitions down to pH approximately 4. The two observed conformational transitions appear to be linked to the titration of the two heme propionate groups, which are suggested to exist in various orientations as a function of both pH and temperature. Heme orientational disorder in the ratio 5:1 was demonstrated by both isotope labeling and NOE experiments. The exchange rate with bulk water of the proximal histidyl labile ring proton is faster in Aplysia than in sperm whale myoglobin, consistent with a greater tendency for local unfolding of the heme pocket in the former protein. A similar increased heme pocket lability in Aplysia myoglobin has been noted in the rate of heme reorientation [Bellelli, A., Foon, R., Ascoli, F.,& Brunori, M. (1987) Biochem. J. 246, 787-789].

Solution 1H NMR Study of the Influence of Distal Hydrogen Bonding and N Terminus Acetylation on the Active Site Electronic and Molecular Structure of Aplysia limacinaCyanomet Myoglobin

“…Heme Orientational Heterogeneity-1 H NMR spectra for both metMb (51) and metMbCN (19,20) had shown that in solution, ϳ20 -25% of the globin possesses a heme rotated 180°about the ␣,␥-meso axis with respect to the unique orientation reported in the crystal structure (12) (shown in Fig. 1).…”

“…Hyperfine shifts reflect electronic and/or magnetic properties of the chromophore, and hence can be extraordinarily sensitive to (and hence render detectable) small structural changes that are unlikely to be detected in either a crystal structure or a solution NMR structure of a diamagnetic analog. Perhaps the best examples of the exquisite sensitivity are the observation of isotope effects on iron-porphyrin covalency due to the distal H-bond to the bound ligand (16 -18) and the characterization of small populations of globins with alternate orientation of the heme about the ␣,␥-meso axis (19,20).…”

“…Hyperfine shifts reflect electronic and/or magnetic properties of the chromophore, and hence can be extraordinarily sensitive to (and hence render detectable) small structural changes that are unlikely to be detected in either a crystal structure or a solution NMR structure of a diamagnetic analog. Perhaps the best examples of the exquisite sensitivity are the observation of isotope effects on iron-porphyrin covalency due to the distal H-bond to the bound ligand (16 -18) and the characterization of small populations of globins with alternate orientation of the heme about the ␣,␥-meso axis (19,20).The information content is particularly rich in the cyanomet globins, in which the bound cyanide can model both the H-bond acceptor properties of bound O 2 (21-23) and the potential steric tilt/bend of bound CO (17,[24][25][26][27][28]. The large dipolar shifts, moreover, guarantee that any heme pocket labile proton can be detected (usually resolved), and its role in H-bonding to the ligand elucidated directly by its placement in the distal pocket (17,22,23,26,28), and indirectly by the expected influence of such a distal H-bond on the electronic structure of the heme (16 -18).…”

Solution of 1H NMR Structure of the Heme Cavity in the Oxygen-avid Myoglobin from the Trematode Paramphistomum epiclitum

“…The Minor Component-Sufficient assignments are pursued in order to establish whether the minor component (ϳ40%) arises from the heme reoriented 180°about the ␣,␥-meso axis (30,31), from another polypeptide chain (isoform), or from damage or modification during isolation. The upfield TOCSY map reveals three minor component methyl peaks (labeled X 2 , X 3 , and X 4 in Fig.…”

Nuclear‐magnetic‐resonance investigation of the cooperative homodimeric hemoglobin from the mollusc Scapharca inaequivalvis

“…Detailed methyl assignments confirm these expectations in all published 'H-NMR spectra For an axial imidazole orientation with its projection bisecting the N-Fe-N vector (C in Fig.l), the methyl contact shift asymmetry is expected to collapse and lead to very similar contact shifts for all four methyls, with the asymmetry dominated by the smaller local peripheral perturbations. Indeed, recent X-ray data have shown that Aplysia Mb possesses an axial histidyl imidazole oriented with a projection passing closer to the meso position than the pyrroles [58] and 'H-NMR resonance assignments of the cyano-met Mb complex reveal strongly reduced electron asymmetry and very similar shifts for the four methyls [59]. Thus the methyl contact shift pattern indicates that the major rhombic perturbation originates on a vector more closely bisecting the N-Fe-N axis rather than along either axis.…”