Recombinant Hemoglobins with Low Oxygen Affinity and High Cooperativity

Ho, Chien; Sun, Dazhen Philip; Shen, Tong-Jian; Ho, Nancy T.; Zou, Ming; Hu, C.K.; Sun, Zhenfan; Lukin, Jonathan A.

doi:10.1016/b978-044420524-7/50023-0

Cited by 11 publications

(23 citation statements)

References 29 publications

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“…We have monitored the occurrence of the T structural marker at 14.2 ppm from DSS in the spectra of our rHbs. As reported earlier, the double mutant, rHb(αV96W, βN108K), shows the T structural marker at 14.2 ppm ( , ). The triple mutant, rHb(αL29F, αV96W, βN108K), also exhibits the T structural marker at 14.2 ppm (Figure ).…”

Section: Resultssupporting

confidence: 55%

“…A unique feature of our mutant Hbs with low oxygen affinity and high cooperativity is the appearance of the 14.2-ppm exchangeable proton resonance on lowering the temperature and/or adding IHP to these rHbs in the carbonmonoxy form ( − ). This resonance has been assigned to the H-bond between α42Tyr and β99Asp located in the α 1 β 2 subunit interface in the T quaternary structure ().…”

Section: Resultsmentioning

confidence: 99%

“…With this expression system, we can design and express any mutant hemoglobins needed for our research. Recently, we have constructed a class of mutant Hbs having low oxygen affinity and high cooperativity ( − ). A unique feature of this class of mutant Hbs is that in their ligated form (such as the carbonmonoxy form), their R (ligated) quaternary structure can be switched to the T form, without changing the ligation state of the Hb molecule, by lowering the temperature and/or by adding an allosteric effector, such as inositol hexaphosphate (IHP), to the Hb solution.…”

mentioning

confidence: 99%

“…A unique feature of this class of mutant Hbs is that in their ligated form (such as the carbonmonoxy form), their R (ligated) quaternary structure can be switched to the T form, without changing the ligation state of the Hb molecule, by lowering the temperature and/or by adding an allosteric effector, such as inositol hexaphosphate (IHP), to the Hb solution. Some of these recombinant hemoglobins (rHbs) exhibit properties making them potential Hb-based oxygen carriers and Hb therapeutics ( − ). rHb(αV96W, βN108K) is one of these novel Hbs, having the lowest oxygen affinity combined with good cooperativity studied so far.…”

mentioning

confidence: 99%

“…Our laboratory has constructed a very low oxygen affinity mutant, rHb(αV96W, βN108K), by combining two mutations, α96Val → Trp (located in the α 1 β 2 subunit interface) and β108Asn → Lys (located in the α 1 β 1 subunit interface and the central cavity) ( , ). This double mutant rHb exhibits very high rates of autoxidation and azide-induced oxidation as expected from the inverse relationship between oxygen affinity and autoxidation discussed above.…”

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confidence: 99%

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Recombinant Hemoglobin(α29Leucine → Phenylalanine, α96Valine → Tryptophan, β108Asparagine → Lysine) Exhibits Low Oxygen Affinity and High Cooperativity Combined with Resistance to Autoxidation

Jeong

Hendrich

et al. 1999

Biochemistry

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Using our hemoglobin expression system in Escherichia coli, we have constructed three recombinant hemoglobins (rHbs) with amino acid substitutions located in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces and in the distal heme pocket of the alpha-chain: rHb(alphaV96W, betaN108K), rHb(alphaL29F, alphaV96W, betaN108K), and rHb(alphaL29F). rHb(alphaV96W, betaN108K) exhibits low oxygen affinity and high cooperativity and also ease of autoxidation of the heme iron atoms from the Fe2+ state to the Fe3+ state. It has been reported by Olson and co-workers [Carver et al., (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that a mutation at position 29 (B10, helix notation), e.g. , Leu --> Phe, can inhibit the autoxidation of the heme iron of myoglobin. We have introduced such a mutation into our rHb having low oxygen affinity and high cooperativity. This triply mutated rHb(alphaL29F, alphaV96W, betaN108K) is stabilized against autoxidation and azide-induced oxidation compared to the double mutant, rHb(alphaV96W, betaN108K), but still exhibits low oxygen affinity and good cooperativity. According to electron paramagnetic resonance results, the oxidized form of the triple mutant shows a high ratio of an anionic form of bishistidine hemichrome. Previous reports have suggested that this form does not have water present at the distal heme pocket. (1)H nuclear magnetic resonance spectra of the triple mutant in the ferric state also exhibit spectral features characteristic of hemichrome-type signals. We have carried out a series of biochemical measurements to characterize these three interesting rHbs and to compare them to human normal adult hemoglobin. These results provide new insights into the structure-function relationship of hemoglobin with amino acid substitutions in the alpha(1)beta(1) and alpha(1)beta(2) interfaces and in the heme pockets.

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Section: Resultssupporting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Recombinant Hemoglobin(α29Leucine → Phenylalanine, α96Valine → Tryptophan, β108Asparagine → Lysine) Exhibits Low Oxygen Affinity and High Cooperativity Combined with Resistance to Autoxidation

Jeong

Hendrich

et al. 1999

Biochemistry

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An Additional H-Bond in the α₁β₂ Interface as the Structural Basis for the Low Oxygen Affinity and High Cooperativity of a Novel Recombinant Hemoglobin (βL105W)

et al. 2000

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Site-directed mutagenesis has been used to construct three recombinant mutant hemoglobins (rHbs), rHb(beta L105W), rHb(alpha D94A/betaL105W), and rHb(alpha D94A). rHb(beta L105W) is designed to form a new hydrogen bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface to lower the oxygen binding affinity by stabilizing the deoxy quaternary structure. We have found that rHb(beta L105W) does indeed possess a very low oxygen affinity and maintains normal cooperativity (P(50) = 28.2 mmHg, n(max) = 2.6 in 0.1 M sodium phosphate at pH 7.4) compared to those of Hb A (P(50) = 9.9 mmHg, n(max) = 3.2 at pH 7.4). rHb(alpha D94A/beta L105W) and rHb(alpha D94A) are expressed to provide evidence that rHb(betaL 105W) does form a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. Our multinuclear, multidimensional nuclear magnetic resonance (NMR) studies on (15)N-labeled rHb(beta L105W) have identified the indole nitrogen-attached (1)H resonance of beta 105Trp for rHb(beta L105W). (1)H NMR studies on Hb A and mutant rHbs have been used to investigate the structural basis for the low O(2) affinity of rHb(beta L105W). Our NMR results provide evidence that rHb(beta L105W) forms a new H-bond from beta 105Trp to alpha 94Asp in the alpha(1)beta(2) subunit interface of the deoxy quaternary structure. The NMR results also show that these three rHbs can switch from the R quaternary structure to the T quaternary structure in their ligated state upon addition of an allosteric effector, inositol hexaphosphate. We propose that the low O(2) affinity of rHb(beta L105W) is due to the formation of a new H-bond between alpha 105Trp and alpha 94Asp in the deoxy quaternary structure.

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Novel Recombinant Hemoglobin, rHb (βN108Q), with Low Oxygen Affinity, High Cooperativity, and Stability against Autoxidation

Tsai

Fang

et al. 2000

Biochemistry

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Using our Escherichia coli expression system, we have constructed rHb (beta N108Q), a new recombinant hemoglobin (rHb), with the amino acid substitution located in the alpha(1)beta(1) subunit interface and in the central cavity of the Hb molecule. rHb (beta N108Q) exhibits low oxygen affinity, high cooperativity, enhanced Bohr effect, and slower rate of autoxidation of the heme iron atoms from the Fe(2+) to the Fe(3+) state than other low-oxygen-affinity rHbs developed in our laboratory, e.g., rHb (alpha V96W) and rHb (alpha V96W, beta N108K). It has been reported by Olson and co-workers [Carver et al. (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that the substitution of phenylalanine for leucine at position 29 of myoglobin can inhibit autoxidation in myoglobin and at position 29 of the alpha-chain of hemoglobin can lower NO reaction in both the deoxy and the oxy forms of human normal adult hemoglobin. Hence, we have further introduced this mutation, alpha L29F, into beta N108Q. rHb (alpha L29F, beta N108Q) is stabilized against auto- and NO-induced oxidation as compared to rHb (beta N108Q), but exhibits lower oxygen affinity at pH below 7.4 and good cooperativity as compared to Hb A. Proton nuclear magnetic resonance (NMR) studies show that rHb (beta N108Q) has similar tertiary structure around the heme pockets and quaternary structure in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces as compared to those of Hb A. The tertiary structure of rHb (alpha L29F, beta N108Q) as measured by (1)H NMR, especially the alpha-chain heme pocket region (both proximal and distal histidyl residues), is different from that of CO- and deoxy-Hb A, due to the amino acid substitution at alpha L29F. (1)H NMR studies also demonstrate that rHb (beta N108Q) can switch from the R quaternary structure to the T quaternary structure without changing ligation state upon adding an allosteric effector, inositol hexaphosphate, and reducing the temperature. On the basis of its low oxygen affinity, high cooperativity, and stability against autoxidation, rHb (beta N108Q) is considered a potential candidate for the Hb-based oxygen carrier in a blood substitute system.

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Recombinant Hemoglobins with Low Oxygen Affinity and High Cooperativity

Cited by 11 publications

References 29 publications

Recombinant Hemoglobin(α29Leucine → Phenylalanine, α96Valine → Tryptophan, β108Asparagine → Lysine) Exhibits Low Oxygen Affinity and High Cooperativity Combined with Resistance to Autoxidation

Recombinant Hemoglobin(α29Leucine → Phenylalanine, α96Valine → Tryptophan, β108Asparagine → Lysine) Exhibits Low Oxygen Affinity and High Cooperativity Combined with Resistance to Autoxidation

An Additional H-Bond in the α₁β₂ Interface as the Structural Basis for the Low Oxygen Affinity and High Cooperativity of a Novel Recombinant Hemoglobin (βL105W)

Novel Recombinant Hemoglobin, rHb (βN108Q), with Low Oxygen Affinity, High Cooperativity, and Stability against Autoxidation

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Recombinant Hemoglobins with Low Oxygen Affinity and High Cooperativity

Cited by 11 publications

References 29 publications

Recombinant Hemoglobin(α29Leucine → Phenylalanine, α96Valine → Tryptophan, β108Asparagine → Lysine) Exhibits Low Oxygen Affinity and High Cooperativity Combined with Resistance to Autoxidation

Recombinant Hemoglobin(α29Leucine → Phenylalanine, α96Valine → Tryptophan, β108Asparagine → Lysine) Exhibits Low Oxygen Affinity and High Cooperativity Combined with Resistance to Autoxidation

An Additional H-Bond in the α1β2 Interface as the Structural Basis for the Low Oxygen Affinity and High Cooperativity of a Novel Recombinant Hemoglobin (βL105W)

Novel Recombinant Hemoglobin, rHb (βN108Q), with Low Oxygen Affinity, High Cooperativity, and Stability against Autoxidation

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An Additional H-Bond in the α₁β₂ Interface as the Structural Basis for the Low Oxygen Affinity and High Cooperativity of a Novel Recombinant Hemoglobin (βL105W)