Cysteines β93 and β112 as Probes of Conformational and Functional Events at the Human Hemoglobin Subunit Interfaces

Vasquez, Gregory B.; Karavitis, Michael; Ji, Xinhua; Pechik, Igor; Brinigar, William S.; Gilliland, Gary L.; Fronticelli, Clara

doi:10.1016/s0006-3495(99)77180-8

Cited by 32 publications

(41 citation statements)

References 37 publications

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“…The effect of these modifications is an increase in microvascular damage and permeability compared with other chemically modified Hbs in rats (16), although it is not yet known whether these specific modifications are responsible for the microvascular toxicity (5). Many such modifications have been shown to influence normal dynamic conversion between R and T states of Hb and heme stability (9,276). It is important to note, however, that some modifications, such as nonspecific modification of bovine Hb with glyceraldehydes to form polyHb, do not appear to destabilize heme binding relative to native Hb, as these do not modify critical amino acids, such as cysteine or methionine residues (16).…”

Section: A the Rational Design Of Hemoglobin-based Oxygen Carriersmentioning

confidence: 99%

The Redox Activity of Hemoglobins: From Physiologic Functions to Pathologic Mechanisms

Reeder

2010

Antioxidants & Redox Signaling

166

184

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Pentacoordinate respiratory hemoproteins such as hemoglobin and myoglobin have evolved to supply cells with oxygen. However, these respiratory heme proteins are also known to function as redox enzymes, reacting with compounds such as nitric oxide and peroxides. The recent discoveries of hexacoordinate hemoglobins in vertebrates and nonsymbiotic plants suggest that the redox activity of globins is inherent to the molecule. The uncontrolled formation of radical species resulting from such redox chemistry on respiratory hemoproteins can lead to oxidative damage and cellular toxicity. In this review, we examine the functions of various globins and the mechanisms by which these globins act as redox enzymes under physiologic conditions. Evidence that redox reactions also occur under disease conditions, leading to pathologic complications, also is examined, focusing on recent discoveries showing that the ferryl oxidation state of these hemoproteins is present in these disease states in vivo. In addition, we review the latest advances in the understanding of globin redox mechanisms and how they might affect cellular signaling pathways and how they might be controlled therapeutically or, in the case of hemoglobin-based blood substitutes, through rational design.

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Section: A the Rational Design Of Hemoglobin-based Oxygen Carriersmentioning

confidence: 99%

The Redox Activity of Hemoglobins: From Physiologic Functions to Pathologic Mechanisms

Reeder

2010

Antioxidants & Redox Signaling

166

184

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“…In the β chain of HbA, the residue Cys F9 undergoes obvious structural changes on ligand binding (see above), and its chemical modification influences O 2 affinity and biases the allosteric transition. Cys F9β was mutated to Ser by Perutz and co-workers [130] and to Ala by Fronticelli and co-workers [131]. Mutation of Cys F9β to Ser causes an increase of the O 2 affinity and a slight decrease of cooperativity, with the Bohr effect being scarcely affected.…”

Section: Mutants Of the Allosteric Corementioning

confidence: 99%

“…Mutation of the same residue to Ala causes a reduction of the Bohr effect, tentatively attributed to the weakening of the important salt bridge between His HC3β 1 and Asp FG1β 1 (see Table 6). Vasquez et al [131] assigned an important role to the empty space formed as a consequence of the substitution of Cys with Ala, which releases the tight packing of the region of the α 1 β 2 interface and allows His HC3β a greater mobility. A further extensive characterization of the role of Cys F9β has been presented by Cheng et al [132], who characterized four Hb mutants (Cys F9β into Gly, Ala, Met and Leu) and compared their properties to those of Hb reacted at Cys F9β with iodoacetamide or Nethyl maleimide.…”

Section: Mutants Of the Allosteric Corementioning

confidence: 99%

“…They found that the mutation Cys G14β→Gly at the α 1 β 1 interface lowers the tendency of Hb to dissociate into dimers by four-fold; since dissociation involves the α 1 β 2 interface, this finding implies that slight distortions of the intra-dimeric contacts are somehow transmitted to the contacts between dimers [131,134].…”

Section: The α 1 β 1 Interfacementioning

confidence: 99%

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The Allosteric Properties of Hemoglobin: Insights from Natural and Site Directed Mutants

Bellelli

Brunori

Miele³

et al. 2006

Current Protein and Peptide Science

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After over a century of extensive research, hemoglobin has become the prototype of allosteric and cooperative proteins. Its molecular structure, known in great detail, has allowed the design of hundreds of site directed mutations, aimed at interfering with its function, and thus at testing our hypotheses on the molecular mechanisms of allostery. The wealth of information thus obtained is difficult to read except for specialists, not only because it makes use of many different technical approaches, but also because of its intrinsically patchy nature. Moreover, several researchers have tried to assign specific roles to segments of the polypeptide chains, rather than to single residues, and have tested their hypotheses by multiple point mutations or by complete replacement with the homologous segment from a different hemoglobin to produce chimeric macromolecules. This approach is in great need of a revision since putative functionally relevant segments partially overlap. This review briefly describes the structure and function of hemoglobin, and analyzes the effect of point mutations, multiple mutations and segment replacement, with special attention to possible biotechnological applications, ranging from pharmacology (Hb solutions as resuscitating fluids and sources of the protein found in hemoglobinopathies for biochemical studies) to bioreactors. Occasional reference is made to site directed mutants of myoglobin, whenever this helps clarifying perplexing results obtained on hemoglobin.

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“…However, the nature of the structural changes responsible for these changes in the oxygen-binding properties is not clear. On the basis of the crystal structure of Hb A in the deoxy state, it has been proposed that the salt bridge between 146His and 94Asp might affect the oxygen-binding properties of Hb modified at the 93 site (16), but no direct evidence has been obtained from Hb in solution to support this hypothesis.…”

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confidence: 99%

Ligand Binding Properties and Structural Studies of Recombinant and Chemically Modified Hemoglobins Altered at β93 Cysteine

et al. 2002

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To investigate the roles of beta93 cysteine in human normal adult hemoglobin (Hb A), we have constructed four recombinant mutant hemoglobins (rHbs), rHb (betaC93G), rHb (betaC93A), rHb (betaC93M), and rHb (betaC93L), and have prepared two chemically modified Hb As, Hb A-IAA and Hb A-NEM, in which the sulfhydryl group at beta93Cys is modified by sulfhydryl reagents, iodoacetamide (IAA) and N-ethylmaleimide (NEM), respectively. These variants at the beta93 position show higher oxygen affinity, lower cooperativity, and reduced Bohr effect relative to Hb A. The response of some of these Hb variants to allosteric effectors, 2,3-bisphosphoglycerate (2,3-BPG) and inositol hexaphosphate (IHP), is decreased relative to that of Hb A. The proton nuclear magnetic resonance (NMR) spectra of these Hb variants show that there is a marked influence on the proximal heme pocket of the beta-chain, whereas the environment of the proximal heme pocket of the alpha-chain remains unchanged as compared to Hb A, suggesting that higher oxygen affinity is likely to be determined by the heme pocket of the beta-chain rather than by that of the alpha-chain. This is further supported by NO titration of these Hbs in the deoxy form. For Hb A, NO binds preferentially to the heme of the alpha-chain relative to that of the beta-chain. In contrast, the feature of preferential binding to the heme of the alpha-chain becomes weaker and even disappears for Hb variants with modifications at beta93Cys. The effects of IHP on these Hbs in the NO form are different from those on HbNO A, as characterized by (1)H NMR spectra of the T-state markers, the exchangeable resonances at 14 and 11 ppm, reflecting that these Hb variants have more stability in the R-state relative to Hb A, especially rHb (betaC93L) and Hb A-NEM in the NO form. The changes of the C2 proton resonances of the surface histidyl residues in these Hb variants in both the deoxy and CO forms, compared with those of Hb A, indicate that a mutation or chemical modification at beta93Cys can result in conformational changes involving several surface histidyl residues, e.g., beta146His and beta2His. The results obtained here offer strong evidence to show that the salt bridge between beta146His and beta94Asp and the binding pocket of allosteric effectors can be affected as the result of modifications at beta93Cys, which result in the destabilization of the T-state and a reduced response of these Hbs to allosteric effectors. We further propose that the impaired alkaline Bohr effect can be attributed to the effect on the contributions of several surface histidyl residues which are altered because of the environmental changes caused by mutations and chemical modifications at beta93Cys.

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Cysteines β93 and β112 as Probes of Conformational and Functional Events at the Human Hemoglobin Subunit Interfaces

Cited by 32 publications

References 37 publications

The Redox Activity of Hemoglobins: From Physiologic Functions to Pathologic Mechanisms

The Redox Activity of Hemoglobins: From Physiologic Functions to Pathologic Mechanisms

The Allosteric Properties of Hemoglobin: Insights from Natural and Site Directed Mutants

Ligand Binding Properties and Structural Studies of Recombinant and Chemically Modified Hemoglobins Altered at β93 Cysteine

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