Hemoprotein models: NMR of imidazole chelated protohemin cyanide complexes

Traylor, Teddy G.; Berzinis, Albin P.

doi:10.1021/ja00528a058

Cited by 70 publications

(48 citation statements)

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“…Structural Differences between WT and rWT-The difference in heme methyl contact shifts between WT and rWT dictates (see under "Discussion") a difference in the orientation of the axial His 95 (F8) imidazole plane relative to the heme (29,30,32,33), with in Fig. 1 predicted to decrease (rotate counterclockwise in Fig.…”

Section: Resultsmentioning

confidence: 99%

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

Nguyen¹,

Xia²,

Cutruzzolà³

et al. 2000

Journal of Biological Chemistry

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The sea hare Aplysia limacina possesses a myoglobin in which a distal H-bond is provided by Arg E10 rather than the common His E7. Solution 1 H NMR studies of the cyanomet complexes of true wild-type (WT), recombinant wild-type (rWT), and the V(E7)H/R(E10)T and V(E7)H mutants of Aplysia Mb designed to mimic the mammalian Mb heme pocket reveal that the distal His in the mutants is rotated out of the heme pocket and is unable to provide a stabilizing H-bond to bound ligand and that WT and rWT differ both in the thermodynamics of heme orientational disorder and in heme contact shift pattern. The mean of the four heme methyl shifts is shown to serve as a sensitive indicator of variations in distal H-bonding among a set of mutant cyanomet globins. The heme pocket perturbations in rWT relative to WT were traced to the absence of the N-terminal acetyl group in rWT that participates in an H-bond to the EF corner in WT. Analysis of dipolar contacts between heme and axial His and between heme and the protein matrix reveal a small ϳ2°rotation of the axial His in rWT relative to true WT and a ϳ3°rotation of the heme in the double mutant relative to rWT Mb. It is demonstrated that both the direction and magnitude of the rotation of the axial His relative to the heme can be determined from the change in the pattern of the contact-dominated heme methyl shift and from the dipolar-dominated heme meso-H shift. However, only NOE data can determine whether it is the His or heme that actually rotates in the protein matrix. Myoglobin (Mb)1 is a member of the globin family of proteins of approximately 140 -150 residues that encapsulate heme and exhibit a remarkably strongly conserved fold of seven to eight helices (A-H) despite a high variability in sequence (1-3). The heme is wedged between the E and F helices, and only the axial (proximal) His F8 (eighth residue on helix F) and Phe CD1 (first residue on the loop between the C and D helices) are completely conserved. This conserved globin fold, however, results in a very wide range of functionality, which appears to be controlled primarily by the nature of the "distal" residues at position E7, E11, E10, and B10 that line the ligand binding pocket (4). The major interaction that strongly stabilizes O 2 over CO binding has been shown to involve H-bonding to the bound O 2 by a distal residue, although destabilization of the bound CO by distal steric interaction cannot be completely discounted (4 -6). Although the distal H-bond in vertebrate globins is always provided by residue E7 (which is overwhelmingly His, but occasionally Gln (2)), there is much more variation in the nature of the E7 residue and the position of the distal H-bond donor in nonvertebrate globins (3). Such alternate residue H-bond stabilization of the bound O 2 has been established in natural globins from sea hares (Arg E10) (7) and trematodes (Tyr B10) (8 -10) and in synthetic globins at position E11 (Asn and Thr) (11).An effective strategy for determining the role of individual residues is to perform functional and m...

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Section: Resultsmentioning

confidence: 99%

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

Nguyen¹,

Xia²,

Cutruzzolà³

et al. 2000

Journal of Biological Chemistry

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“…52 We have determined the NMR of chelated hemin cyanides in which the imidazole rotational angle is changed and the rotational freedom reduced by changing from a free rotating external 1-methylimidazole base to a flexible chelated heme with -(CH2)4~linkage ( = 4, G = NH, Y = vinyl, B = 1-imidazolyl in Figure 3 to a less flexible chelated heme with a -(CH2)3~linkage ( = 3). 53 The methyl group resonances change in this series from a spread of 5 ppm in the external base hemin cyanide complex to 7 ppm in the = 4 chelated protoheme to 17 ppm in the more rigid = 3 chelated protoheme. The n = 3 chelated protoheme spectrum resembles that of hemoglobin+-CN'.…”

Section: Structure-reactivitymentioning

confidence: 85%

Synthetic model compounds for hemoproteins

Traylor¹

1981

Acc. Chem. Res.

171

103

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“…Ser 92 Has Little Effect on Im Conformation. The hyperfine shifts in the 1 H NMR spectra of MbCN complexes are very sensitive to changes in heme axial ligand conformation, and comparisons of these shifts in different mutants can be used to assess changes in conformation of heme ligands and the local amino acid environment (5,13,(15)(16)(17). For example, this pattern is very different in WT MbCN and H93G(Im)CN (5).…”

Section: Discussionmentioning

confidence: 99%

“…1 H NMR spectroscopy of the metcyano complexes provides a great deal of information about the structure and dynamics of these mutants. MbCN is a paramagnetic (S ) 1 / 2 ) species; the hyperfine shifts of protons of the heme and amino acids of the heme pocket are very sensitive to changes in conformation of the proximal ligand (15)(16)(17). Each new exogenous ligand in H93G gives a unique fingerprint hyperfine shifted region of the MbCN 1 H NMR spectrum (2).…”

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confidence: 99%

Hydrogen Bonding Modulates Binding of Exogenous Ligands in a Myoglobin Proximal Cavity Mutant

et al. 1999

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In the sperm whale myoglobin mutant H93G, the proximal histidine is replaced by glycine, leaving a cavity in which exogenous imidazole can bind and ligate the heme iron (Barrick, D. (1994) Biochemistry 33, 6545-6554). Structural studies of this mutant suggest that serine 92 may play an important role in imidazole binding by serving as a hydrogen bond acceptor. Serine 92 is highly conserved in myoglobins, forming a well-characterized weak hydrogen bond with the proximal histidine in the native protein. We have probed the importance of this hydrogen bond through studies of the double mutants S92A/H93G and S92T/H93G incorporating exogenous imidazole and methylimidazoles. 1 H NMR spectra reveal that loss of the hydrogen bond in S92A/H93G does not affect the conformation of the bound imidazole. However, the binding constants for imidazoles to the ferrous nitrosyl complex of S92A/H93G are much weaker than in H93G. These results are discussed in terms of hydrogen bonding and steric packing within the proximal cavity. The results also highlight the importance of the trans diatomic ligand in altering the binding and sensitivity to perturbation of the ligand in the proximal cavity.Exogenous molecules which serve a specific structural or functional role can be inserted into engineered protein cavities as an extension of site-directed mutagenesis for dissecting protein structure-function relationships in heme proteins (1-4). When a residue which ligates the heme iron (such as histidine) is mutated to a smaller, nonligating amino acid (typically alanine or glycine), exogenous molecules which may mimic natural amino acids side chains or bear nonnatural functionality can be introduced into the resulting cavity and incorporated into the protein structure. For example, in sperm whale myoglobin (Mb 1 ), the proximal histidine (His 93) is the sole covalent linkage between the polypeptide chain and the heme iron. In the mutant H93G, this residue has been replaced by glycine, creating a cavity in the proximal heme pocket. When H93G Mb is expressed in Escherichia coli in the presence of imidazole, the protein is isolated with an imidazole molecule occupying that cavity and serving as an axial ligand to the heme iron (1). This imidazole can be replaced with a variety of exogenous ligands via a simple dialysis technique, producing protein complexes that are distinguishable by many physical observables (2, 5-7). These proteins are denoted H93G(L), where L represents the bound proximal ligand, and they are hybrids of model compounds and site-directed mutants. As in model compounds, the heme ligand can be systematically varied beyond the limited range of the naturally occurring amino acids, but because the protein architecture is preserved, interactions between the polypeptide and the heme ligand can also be studied.The most striking difference between the structures of metaquoH93G(Im) and wild-type myoglobin (WTMb) is in the conformation of the proximal imidazole ring. The exogenous proximal imidazole of H93G(Im) is rotated ab...

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Hemoprotein models: NMR of imidazole chelated protohemin cyanide complexes

Cited by 70 publications

References 2 publications

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

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

Synthetic model compounds for hemoproteins

Hydrogen Bonding Modulates Binding of Exogenous Ligands in a Myoglobin Proximal Cavity Mutant

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