Haem disorder in two myoglobins: comparison of reorientation rate

Abstract

The globins from sperm whale and from Aplysia limacina myoglobins were reconstituted by addition of stoichiometric ferric protohaem and the Soret c.d. was followed as a function of time. For both reconstituted proteins, the Soret c.d. changes with time, reflecting haem reorientation inside its pocket, as previously described [Aojula, Wilson & Drake (1986) Biochem. J. 237,[613][614][615][616] for sperm whale myoglobin. The time course of the c.d. transition is found to be approx. 10 times faster in Aplysia than in sperm whale myoglobin, a result which is in agreement with the known structural and physicochemical properties of the two myoglobins; furthermore, these results confirm that c.d. and n.m.r. data on haem orientation in haemoproteins reflect the same molecular phenomenon. INTRODUCTIONSeveral haemoproteins have been described to exist in two interconvertible conformations, differing in their n.m.r. properties. La Mar and collaborators (La Mar et al., 1978) have shown that the coexistence of conformers is related to the fact that the intrinsically asymmetric haem molecule can assume two orientations differing by 180°rotation around its a-y axis. Often one of the conformers is largely prevalent at equilibrium, and constitutes the 'native' (or ordered) conformation as detected by X-ray crystallography. In the case of sperm whale myoglobin the disordered form accounts for only 10% of the total haemoprotein in solution (La Mar et al., 1983), while in other cases the disordered form can be as populated as the ordered one, as in tuna fish myoglobin (Levy et al., 1985).Immediately after reconstitution obtained by mixing sperm whale apomyoglobin with either ferrous or ferric haem, the disordered form accounts for approx. 50 % of the total pigment; this population ratio changes with time and decays slowly towards its equilibrium value. The re-equilibration has been followed by means of n.m.r. (La Mar et al., 1984) or c.d. spectroscopy (Aojula et al., 1986) and the measured half-time ranges from hours to days depending on the experimental conditions (for example, pH and temperature).To a first approximation it may be assumed that the prevalent pathway to achieve haem reorientation requires dissociation of the porphyrin from the protein, since the haem pocket appears to be too small to allow the haem to rotate freely; for this reason we compared the rate of haem reorientation in two myoglobins whose physicochemical properties, particularly with respect to haem affinity, differ widely: those from sperm whale and from Aplysia limacina; in fact myoglobin from Aplysia limacina

Properties of a recombinant human hemoglobin with aspartic acid 99 (β), an important intersubunit contact site, substituted by lysine

“…Indeed, most mammalian Hb are tetrameric, enabling them to interact efficiently with allosteric regulators. However, some dimeric Hb in the unliganded form, i.e., from the mollusc bivalve clams, show very similar negative and positive ellipticity in the region of the Soret band as found for the D99K(P)-1 Hb (Chiancone et al, 1981(Chiancone et al, , 1990Antonini et al, 1984;Bellelli et al, 1987). The interaction of the heme with 2 different environments of the adjacent globin may be responsible for generation of the D99K(/3)-1 and D99K(P)-2 forms.…”

Haem-binding-site heterogeneity and haem Cotton effects of Glycera dibranchiata monomeric haemoglobins

“…Previous studies of various myoglobins have suggested a relationship between the magnitude of the c.d. signal and the percentage haem disorder (Bellelli et al, 1987;Light et al, 1987). Here we compare g values, since minor differences in absorption coefficients among the components may exist.…”

The impact of altered protein‐heme interactions on the resonance Raman spectra of heme proteins. Studies of heme rotational disorder

“…Interestingly, this minor conformer has also been reported to exist in the native proteins; e.g., sperm whale, equine, bovine and elephant myoglobins (Mb) show ∼10% of this minor component,7 while human hemoglobin (Hb A) exhibits ∼10% disoriented form in the β subunits, but only ∼2% in the α subunits 10. While this heme reorientation in reconstituted Mbs has also been studied by circular dichroism,14, 15 NMR spectroscopy remains the principal tool for detecting and quantifying heterogeneity in heme containing proteins 5–13…”