Substitution of the Heme Binding Module in Hemoglobin α- and β-Subunits

Inaba, Kenji; Ishimori, Koichiro; Imai, Katsunori; Morishima, Isao

doi:10.1074/jbc.275.17.12438

Cited by 10 publications

(11 citation statements)

References 66 publications

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“…Our gelchromatography results of β 116HisfAsp chains like β 112CysfAsp chains also showed monomer formation predominated (5). The R chains are known to favor formation of monomers rather than tetramers, and NMR resonance signals of the heme region in these chains differ from those of β chains (14,26,29). This difference was thought to be mainly attributed to local structural differences between R and β chains (26).…”

Section: Discussionmentioning

confidence: 57%

Significance of β116 His (G18) at α1β1 Contact Sites for αβ Assembly and Autoxidation of Hemoglobin

et al. 2003

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The role of heterotetramer interaction sites in assembly and autoxidation of hemoglobin is not clear. The importance of beta(116His) (G-18) and gamma(116Ile) at one of the alpha1beta1 or alpha1gamma1 interaction sites for homo-dimer formation and assembly in vitro of beta and gamma chains, respectively, with alpha chains to form human Hb A and Hb F was assessed using recombinant beta(116His)(-->)(Asp), beta(116His)(-->)(Ile), and beta(112Cys)(-->)(Thr,116His)(-->)(Ile) chains. Even though beta chains (e.g., 116 His) are in monomer/tetramer equilibrium, beta(116Asp) chains showed only monomer formation. In contrast, beta(116Ile) and beta(112Thr,116Ile) chains showed homodimer and homotetramer formation like gamma-globin chains which contain 116 Ile. Assembly rates in vitro of beta(116Ile) or beta(112Thr,116Ile) chains with alpha chains were 340-fold slower, while beta(116Asp) chains promoted assembly compared to normal beta-globin chains. These results indicate that amino acid hydrophobicity at the G-18 position in non-alpha chains plays a key role in homotetramer, dimer, and monomer formation, which in turn plays a critical role in assembly with alpha chains to form Hb A and Hb F. These results also suggest that stable dimer formation of gamma-globin chains must not occur in vivo, since this would inhibit association with alpha chains to form Hb F. The role of beta(116His) (G-18) in heterotetramer-induced stabilization of the bond with oxygen in hemoglobin was also assessed by evaluating autoxidation rates using recombinant Hb tetramers containing these variant globin chains. Autoxidation rates of alpha(2)beta(2)(116Asp) and alpha(2)beta(2)(116Ile) tetramers showed biphasic kinetics with the faster rate due to alpha chain oxidation and the slower to the beta chain variants whose rates were 1.5-fold faster than that of normal beta-globin chains. In addition, NMR spectra of the heme area of these two hemoglobin variant tetramers showed similar resonance peaks, which are different from those of Hb A. Oxygen-binding properties of alpha(2)beta(2)(116His)(-->)(Asp) and alpha(2)beta(2)(116His)(-->)(Ile), however, showed slight alteration compared to Hb A. These results suggest that the beta116 amino acid (G18) plays a critical role in not only stabilizing alpha1beta1 interactions but also in inhibiting hemoglobin oxidation. However, stabilization of the bonds between oxygen and heme may not be dependent on stabilization of alpha1beta1 interactions. Tertiary structural changes may lead to changes in the heme region in beta chains after assembly with alpha chains, which could influence stability of dioxygen binding of beta chains.

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Section: Discussionmentioning

confidence: 57%

Significance of β116 His (G18) at α1β1 Contact Sites for αβ Assembly and Autoxidation of Hemoglobin

et al. 2003

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“…Once the methodology and its effectiveness have been established, other possible future work will be to change the size and number of blocks to be deleted or inserted in addition to changing the location where such mutations are introduced. This will be equivalent to a high‐throughput version of the previous efforts performed on nested deletion [17,18] or shuffling of proteins such as hemoglobin [19]. These works are useful not only from a molecular engineering viewpoint (such as creating minimum‐sized GFP) but also from a protein science viewpoint (such as exploring the protein sequence space).…”

Section: Resultsmentioning

confidence: 99%

GFPs of insertion mutation generated by molecular size‐altering block shuffling

2003

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Insertion and deletion analyses of a protein have been less common than point mutation analyses, partly due to the lack in e¡ective methods. This is the case with the green £uo-rescent protein (GFP), which is so widely applied in molecular biology and other ¢elds. In this paper we ¢rst introduce a systematic approach for generating insertion/deletion mutants of GFP. A new technology of Y-ligation-based block shu¥ing (YLBS) was successfully applied to produce size-altered GFPs, providing insertion-containing GFPs of £uorescence, though no deletion type of £uorescence was obtained so far as examined. The analysis of these proteins suggested that size alteration (deletion/insertion) is acceptable so far as some type of rearrangement in a local structure can accommodate it. This paper demonstrates that YLBS can generate insertion and deletion mutant libraries systematically, which are bene¢cial in the study of structure^function relationship. ß

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“…Additionally, studies with bis-histidine Hbs have shown that the cyanide ligand displaces the His-E7 and not the His-F8 bond, which would indicate that the His-F8 bond is stronger than the His-E7 bond 51,52 . Finally, 1 HMR studies of rHbCN and heme-binding modules have shown that DCNh binds to His-F8 and not His-E7 53–55 .…”

Section: Introductionmentioning

confidence: 97%

“…The affinity of heme for the His-F8 residue has been shown to be a major factor regulating association between the heme and globin chain . Additionally, mutated Hbs lacking the His-F8 residue were shown to exhibit much lower heme-binding affinity, faster heme loss, or the absence of heme. − Furthermore, studies with bis-histidine Hbs have shown that the cyanide ligand displaces the His-E7 and not the His-F8 bond, which would indicate that the His-F8 bond is stronger than the His-E7 bond. , Finally, 1 H nuclear magnetic resonance studies of rHbCN and heme-binding modules have shown that DCNh binds to His-F8 and not His-E7. − …”

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confidence: 99%

Quantification of Active Apohemoglobin Heme-Binding Sites via Dicyanohemin Incorporation

2017

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Apohemoglobin (apoHb) is produced by removing heme from hemoglobin (Hb). However, preparations of apoHb may contain damaged globins, which render total protein assays inaccurate for active apoHb quantification. Fortunately, apoHb heme-binding sites react with heme via the proximal histidine-F8 (His-F8) residue, which can be monitored spectrophotometrically. The bond between the His-F8 residue of apoHb and heme is vital for maintenance of fully functional and cooperative Hb. Additionally, most apoHb drug delivery applications facilitate hydrophobic drug incorporation inside the apoHb hydrophobic heme-binding pocket in which the His-F8 residue resides. This makes the His-F8 residue a proper target for apoHb activity quantification. In this work, dicyanohemin (DCNh), a stable monomeric porphyrin species, was used as a probe molecule to quantify active apoHb through monocyanohemin-His-F8 bond formation. ApoHb activity was quantified via the analysis of the 420 nm equilibrium absorbance of DCNh and apoHb mixtures. His-F8 saturation was determined by the presence of an inflection point from a plot of the 420 nm absorbance of a fixed concentration of apoHb against an increasing DCNh concentration. Various concentrations of a stock apoHb solution were tested to demonstrate the precision of the assay. The accuracy of the assay was assessed via spectral deconvolution, confirming His-F8 saturation at the inflection point. The effect of the heme-binding protein bovine serum albumin and precipitated apoHb on assay sensitivity was not significant. An analysis of the biophysical properties of reconstituted Hb confirmed heme-binding pocket activity. Taken together, this assay provides a simple and reliable method for determination of apoHb activity.

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Substitution of the Heme Binding Module in Hemoglobin α- and β-Subunits

Abstract: In our previous work, we demonstrated that the replacement of the "heme binding module," a segment from F1 to G5 site, in myoglobin with that of hemoglobin ␣-subunit converted the heme proximal structure of myoglobin into the ␣-subunit type (

Cited by 10 publications

References 66 publications

Significance of β116 His (G18) at α1β1 Contact Sites for αβ Assembly and Autoxidation of Hemoglobin

Significance of β116 His (G18) at α1β1 Contact Sites for αβ Assembly and Autoxidation of Hemoglobin

GFPs of insertion mutation generated by molecular size‐altering block shuffling

Quantification of Active Apohemoglobin Heme-Binding Sites via Dicyanohemin Incorporation

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