Daniel W. Parish

Heme orientational disorder in reconstituted and native sperm whale myoglobin: Proton nuclear magnetic resonance characterizations by heme methyl deuterium labeling in the met-cyano protein

The solution proton nuclear magnetic resonance spectrum of the Met-cyano form of sperm whale myoglobin reveals the presence of two sets of comparably intense resonances immediately after reacting the apoprotein with hemin, only one of which corresponds to that of the accepted native protein. Isotope labeling of individual methyl groups of hemin reveals that the methyl assignments differ characteristically in that similar resonance positions for the two components arise from the methyl groups related by a 180 degrees rotation about the alpha-gamma-meso axis. This phenomenon, observed earlier only for myoglobin with modified hemin, dictates that the second protein component in solution immediately after reconstitution must have the heme rotated by 180 degrees about the alpha-gamma-meso axis as compared to that found in the single crystal. The two components in the reconstituted protein equilibrate to yield the spectrum of the native Met-cyanomyoglobin for which there still exists approximately 8% of the minor component. Thus native myoglobin in solution is structurally heterogeneous in the heme pocket. Proton nuclear magnetic resonance spectra of deoxymyoglobin produced from both native and freshly reconstituted protein shown that the heterogeneity is also a property of the physiologically relevant reduced protein forms. It is suggested that, contrary to available X-ray data, heme orientational heterogeneity may be the rule rather than the exception in b-type hemoproteins, and that such disorder must be carefully considered in detailed correlations between structure and function even in native hemoproteins.

1H-NMR study of the mechanism of assembly and equilibrium heme orientation of sperm whale myoglobin reconstituted with protohemin type-isomers

The products of the incorporation of various protohemin type-isomers into the heme pocket of sperm whale myoglobin were investigated by 1H-NMR in the met-cyano complexes, both immediately after reconstitution as well as at equilibrium. The type-isomers studied include those involving all possible interchanges of the two substituents on a given pyrrole. The protohemin-III and -XIII isomers, with true 2-fold symmetry, yielded only homogeneous products. Protohemins-XI, -XIV both exhibited two species after reconstitution, with one disappearing with time. Protohemin-I was the only asymmetric hemin that failed to exhibit two isomers initially. The orientation of the hemin within the pocket was established by nuclear Overhauser detected dipolar connectivities among heme substituents and between heme substituents and assigned heme pocket residues. At equilibrium, the heme orientations were dominated by the asymmetric propionate rather than vinyl dispositions on the hemin, with a clear preference for placing a propionate at the 8- vs. 5-methyl position of native myoglobin. For protohemin-XI, the propionates were found in the unexpected positions of the 7-propionate and 2-vinyl groups of native myoglobin, indicating that propionates can occupy positions well within the hydrophobic interior. The alternate heme orientation for the metastable intermediates detected for protohemin-XI and -XIV involved rotational isomerism about the alpha,gamma-meso axes bisecting the vinyl positions, but these two axes are at right angles to each other in the protein matrix. The fact that protohemin-XIV, but not protohemin-I, exhibits a reversed orientation as a reconstitution intermediate provides direct evidence that vinyl contacts, as well as propionate links, modulate the relative stabilities of the initial encounter complexes between hemin and apomyoglobin. The heme cavity molecular/electronic structure was found largely unperturbed for the complexes of the various protohemin type-isomers.

The influence of side chain modifications of the heme moiety on prosthetic acceptance and function of rat hepatic cytochrome P-450 and tryptophan pyrrolase

The relative potential of various structural isomers (III, XIII) and various 2,4-side chain modified analogs of heme (iron-protoporphyrin IX) to incorporate into rat liver hemoproteins, cytochrome P-450(s), and tryptophan pyrrolase was examined. Such assessments for hepatic cytochrome P-450 relied on generation of reconstitutible apocytochrome(s) P-450 by suicidal alkylation of the existing prosthetic heme moiety by allylisopropylacetamide (AIA) in vivo. Subsequent replacement of the prosthetic heme was brought about by incubating the apocytochrome(s) P-450-enriched preparations with a particular heme isomer or analog. Structure-function relationships of the reconstituted isozymes were assessed in microsomal preparations by monitoring cytochrome P-450 content (structure) and its mixed function oxidase activity (function). In parallel, the relative ability of these heme isomers and analogs to functionally constitute hepatic tryptophan pyrrolase was also assessed by monitoring the relative increase in holoenzyme activity when preparations deliberately enriched in constitutible apoenzyme were incubated with each of these compounds. The findings reveal that 2,4-side chain modifications on the heme IX skeleton markedly influence the function of the constituted hemoproteins possibly by affecting their structural assembly through steric, electronic, and/or hydrophobic interactions with the corresponding apoproteins. Furthermore, these studies not only reveal that the structural specifications of the active prosthetic site of rat liver cytochrome P-450(s) differ from those of tryptophan pyrrolase, but also that the structural specifications of these mammalian hemoproteins for their prosthetic heme differ considerably from those reported for their bacterial counterparts.

Proton NMR study of the influence of heme vinyl groups on the formation of the isomeric forms of sulfmyoglobin

The formation of sulfmyoglobin has been investigated for myoglobin reconstituted with hemins having vinyls replaced by hydrogens to determine the participation of the vinyl groups in the reaction processes. Green complexes are produced in all cases, proving that vinyls are not obligatory for the formation of sulfproteins. In the presence of the 4‐vinyl group, the 1H NMR spectra of the met‐cyano derivatives indicate the formation of three green species; however, the most stable of these products is not formed in the absence of this group, confirming reaction of the 4‐vinyl in this species. Two new red extractable sulfmyoglobin derivatives are formed in the absence of the 4‐vinyl group.

Horse heart myoglobin reconstituted with a symmetrical heme A circular dichroism study

Proton NMR studies on myoglobins and hemoglobins reconstituted with non-natural hemes, possessing different side chains in the pyrrolic rings, have provided interesting information for the understanding of the mechanism governing heme reorientation in the globin pocket, during synthesis of the native protein in vivo or in the reconstitution process in vitro. More recently, circular dichroism (CD) studies have been reported as a qualitative, alternative tool, with respect to 1H-NMR for detecting heme disorder in a reconstituted myoglobin or hemoglobin. In this paper, a CD study is reported on the reconstitution of horse heart myoglobin with protoheme XIII, a heme possessing true rotational symmetry about its alpha, gamma-meso axis. The results obtained show that the reconstitution product with this heme, which binds to the apoprotein with high affinity, not dissimilar from that of the natural heme, is characterized by a CD spectrum with bands possessing rotational strengths much lower than in the native protein. Furthermore, the CD changes detected as a function of time, during heme reorientation, in the case of natural heme, are absent when the apoprotein is reconstituted with protoheme XIII. These data provide independent evidence for reorientation of the natural heme, which follows its insertion into the protein matrix.