Electrostatic Modification of the Active Site of Myoglobin:  Characterization of the Proximal Ser92Asp Variant

Lloyd, E. A.; Burk, David L.; Ferrer, Juan C.; Maurus, R.; Doran, J.; Carey, Paul R.; Brayer, G.D.; Mauk, A. Grant

doi:10.1021/bi9608976

Cited by 43 publications

(59 citation statements)

References 88 publications

(97 reference statements)

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“…On the other hand, the M86E mutant does not form a structure with the negative charge of glutamate buried inside the protein. Instead, this glutamate rearranges toward the solvent much the same way that aspartate positions in the S92D mutations of myoglobin (25, 26). The subtle difference between the structures is dependent upon the ability of the carboxyl group to orient so that it can simultaneously interact with the (H89)N δ H and with solvent, in M86D.…”

Section: Discussionmentioning

confidence: 99%

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

et al. 2011

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The proximal side of dehaloperoxidase-hemoglobin A (DHP A) from Amphitrite ornata has been modified via site-directed mutagenesis of methionine-86 into aspartate (M86D) to introduce an Asp-His-Fe triad charge relay. X-ray crystallographic structure determination of the metcyano forms of M86D (PDB 3MYN) and M86E (PDB 3MYM) mutants reveals the structural origins of a stable catalytic triad in DHP A. A decrease in rate of H2O2 activation, as well as a lowered reduction potential than the wild-type enzyme was observed in M86D. One possible explanation for the significantly lower activity is an increased affinity for the distal histidine to bind to the heme Fe to form a bis-histidine adduct. Resonance Raman spectroscopy demonstrates a pH-dependent ligation by the distal histidine in M86D, which is indicative of an increased trans effect. At pH 5.0, the heme Fe is 5-coordinate and this resembles the wild-type DHP A resting state. However, at pH 7.0, the distal histidine appears to form a 6-coordinate ferric bis-histidine (hemichrome) adduct. These observations can be explained by the effect of the increased positive charge on the heme Fe on the formation of a 6-coordinate low-spin adduct, which inhibits the ligation and activation of H2O2 as required for peroxidase activity. The results suggest that the role of the proximal charge relay in peroxidases regulates the redox potential of the heme Fe, but that the trans effect is a carefully balanced property that can both activate H2O2 and attract ligation by the distal histidine. To understand the balance of forces that modulate peroxidase reactivity, three mutants in the M86 position, M86A, M86D, and M86E were studied by spectroelectrochemistry and NMR spectroscopy of 13C15N -labeled cyanide adducts as probes of the redox potential and of the trans effect in the heme Fe, both of which can be correlated with the proximity of negative charge to the Nδ hydrogen of the proximal histidine, consistent with an Asp-His-Fe charge relay observed in heme peroxidases.

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Section: Discussionmentioning

confidence: 99%

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

et al. 2011

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“…Freshly prepared Mb-H appears to be present in solution as a mixture of four isomers, two in a high-spin state and two in a low-spin state. The high-spin isomers are characterized by the peaks at about 53 and 72 ppm, attributable to heme methyls at position 1 and 3, respectively, which are split in two components, and by the four main peaks between 80 and 92 ppm, where the resonances of the heme methyl groups at position 5 and 8 of Mb occur (45). However, the most prominent features occur in the intermediate field region of the spectrum were a couple of intense peaks at 30.5 and 45.5 ppm and a couple of other peaks of notable intensity at 37.5 and 30 ppm are observed.…”

Section: Synthesis Of Modified Hemins and Reconstitution Of Apomyoglomentioning

confidence: 99%

Properties and Reactivity of Myoglobin Reconstituted with Chemically Modified Protohemin Complexes

et al. 2000

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The synthetic complexes protohemin-6(7)-L-arginyl-L-alanine (HM-RA) and protohemin-6(7)-L-histidine methyl ester (HM-H) were prepared by condensation of suitably protected Arg-Ala or His residues with protohemin IX. HM-RA and HM-H were used for reconstitution of apomyoglobin from horse heart, yielding the Mb-RA and Mb-H derivatives, respectively, of the protein. The spectral, binding and catalytic properties of Mb-RA and Mb-H are significantly different from those of Mb. As shown by MM and MD calculations, these differences are determined by some local structural changes around the heme which are generated by increased mobility of a key peptide segment (Phe43-Lys47), containing the residue (Lys45) that in native Mb interacts with one of the porphyrin carboxylate groups. In the reconstituted Mbs this carboxylate group is bound to the Arg-Ala or His residue and is no longer available for electrostatic interaction with Lys45. The mobility of the peptide segment near the active site allows the distal histidine to come to a closer contact with the heme, and in fact Mb-RA and Mb-H exist as an equilibrium between a high-spin form and a major low-spin, six-coordinated form containing a bis-imidazole ligated heme. The two forms are clearly distinguishable in the NMR spectra, that also show that each of them consists of a mixture of the two most stable isomers resulting from cofactor reconstitution, as also anticipated by MM and MD calculations. Exogenous ligands such as cyanide, azide, or hydrogen peroxide can displace the bound distal histidine, but their affinity is reduced. On the other hand, mobilization of the peptide chain around the heme in the reconstituted Mbs increases the accessibility of large donor molecules at the heme periphery, with respect to native Mb, where a rigid backbone limits access to the distal pocket. The increased active site accessibility of Mb-RA and Mb-H facilitates the binding and electron transfer of phenolic substrates in peroxidase-type oxidations catalyzed by the reconstituted proteins in the presence of hydrogen peroxide.

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“…In human Mb, ligand binding is unaffected by F7 replacement, and the orientation of the proximal histidine rotates from the eclipsed position by a few degrees (27). Slight rotation of the proximal histidine is also reported for the S92D variant of horse heart Mb in the cyanomet state (28). The role of proximal interactions was reinspected by Peterson and coworkers, who studied variants containing multiple replacements and 8-residue F-helix swaps (56).…”

Section: Discussionmentioning

confidence: 93%

Proximal Influences in Two-on-Two Globins: Effect of the Ala69Ser Replacement on Synechocystis sp. PCC 6803 Hemoglobin

Knappenberger

Kuriakose

et al. 2006

Biochemistry

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The cyanobacterium Synechocystis sp. PCC 6803 (S6803) expresses a two-on-two globin in which His46 (distal side) and His70 (proximal) function as heme iron axial ligands. His46 can be displaced by O 2 , CO, and CN − , among others, whereas His70 is not labile under native conditions. The residue preceding the proximal histidine has been implicated in controlling globin axial ligand reactivity; the details of the mechanism, however, are not well understood, and little information exists for bis-histidyl hexacoordinate proteins. In many vertebrate hemoglobins and in the Synechocystis protein, the position is occupied by an alanine whereas, in myoglobins, it is a serine involved in an intricate hydrogen bond network. We examined the role of Ala69 in S6803 hemoglobin through the effects of an Ala → Ser replacement. The substitution resulted in minor structural perturbations, but the holoprotein's response to temperature-, urea-, and acid-induced denaturation was measurably affected. Enhanced three-state behavior was manifested in the decoupling of heme binding and secondary structure formation. Urea-gradient gel experiments revealed that the stability of the apoprotein was unchanged by the replacement and that a slight alteration of the folding kinetics occurred in the holoproteins. Cyanide-binding experiments were performed to assess trans effects. The apparent rate constant for association decreased two-fold upon Ala69Ser replacement. This deceleration was attributed to a change in the lifetime of a state containing a decoordinated His46. The results demonstrated that, as in vertebrate globins and leghemoglobin, proximal influences operate to determine fundamental dynamic and thermodynamic properties of the protein.Keywords truncated hemoglobin; 2-on-2 globin; hexacoordinate hemoglobin; urea-gradient gel; heme binding; thermodynamic stabilityIron protoporphyrin IX (Fe-PPIX) 1 serves as a cofactor in a large number of essential proteins. In the absence of covalent attachment to the protein matrix via protoporphyrin substituents, the contact between Fe(II)-PPIX (or b heme) and the protein is limited to ligation bond(s) to the iron, hydrogen bonding, van der Waals and electrostatic interactions, and hydrophobic † This study was supported by National Science Foundation grants MCB-091182 and MCB-0349409, National Institutes of Health grant GM-054217, and NASA grant NNG04GN33H (DAV).* To whom correspondence should be addressed. Tel: (814) NIH Public Access Author ManuscriptBiochemistry. Author manuscript; available in PMC 2008 September 11. forces. The affinity for the heme group and its chemical properties, such as redox potential, ability to bind various ligands, and propensity for generating reactive oxygen species, are strictly controlled by the protein environment.In many b hemoproteins the iron is endogenously hexacoordinated, with the axial positions of its octahedral geometry filled by two histidine side chains (1). A well-known example is cytochrome b 5 , an electron-transfer protein that does not bind...

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Electrostatic Modification of the Active Site of Myoglobin: Characterization of the Proximal Ser92Asp Variant

Cited by 43 publications

References 88 publications

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

Functional Consequences of the Creation of an Asp-His-Fe Triad in a 3/3 Globin

Properties and Reactivity of Myoglobin Reconstituted with Chemically Modified Protohemin Complexes

Proximal Influences in Two-on-Two Globins: Effect of the Ala69Ser Replacement on Synechocystis sp. PCC 6803 Hemoglobin

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