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Kehinde O. Okonjo - One of the best experts on this subject based on the ideXlab platform.
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Bohr Effect and oxygen affinity of carp, eel and human hemoglobin: Quantitative analyses provide rationale for the Root Effect.
Biophysical chemistry, 2018Co-Authors: Kehinde O. OkonjoAbstract:Abstract The functional properties of most fish hemoglobins are more complex than those of human hemoglobin. This complexity arises in the form of the Root Effect, in which the oxygen affinity of such fish hemoglobins decreases rapidly with pH relative to that of human hemoglobin. Cooperative ligand binding is also diminished below pH ≈ 6.5. The Bohr Effect, determined by acid-base titration, has been reported for the Root Effect carp and anodic eel hemoglobins. Unlike for mammalian hemoglobins, the Wyman equation for the Bohr Effect fails to account quantitatively for these Bohr data. We present a successful quantitative accounting for these data based on evidence for multiple T states in various fish hemoglobins and on their lack of sixhistidine Bohr groups, with pKoxy > pKdeoxy. On the same bases we also provide a rationale for the higher pH sensitivity of the oxygen affinity of carp compared to human hemoglobin.
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Bohr Effect of human hemoglobin separation of tertiary and quaternary contributions based on the wyman equation
Biophysical Chemistry, 2017Co-Authors: Kehinde O. OkonjoAbstract:As a prelude to separating tertiary from quaternary structure contributions to the Bohr Effect, we employed the Wyman equation to analyze Bohr data for human hemoglobin to which 2,3-bisphosphoglycerate, 2,3-BPG, is bound. Changes in the pKas of the histidine Bohr groups result in a net reduction of their contributions to the Bohr Effect at pH 7.4 compared to their contributions in stripped hemoglobin. The non-histidine 2,3-BPG binding groups - the β-chain terminal amino group and Lys82β - make negative and positive contributions, respectively, to the Bohr Effect. The final result is that the Bohr Effect at physiological pH is higher for 2,3-BPG bound compared to stripped hemoglobin. Contributions linked to His2β, His77β and His143β enable us to separate tertiary from quaternary Bohr contributions in stripped and in 2,3-BPG bound hemoglobin. Both contributions serve to make the Bohr Effect for 2,3-BPG bound hemoglobin higher than for stripped hemoglobin at physiological pH.
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Bohr Effect of native and chemically modified hemoglobins quantitative analyses based on the wyman equation
Biophysical Chemistry, 2017Co-Authors: Kehinde O. OkonjoAbstract:Thirteen histidines and the α-chain terminal amino group (ACTA) make all of the contributions to the Bohr Effect of human hemoglobin. The pKas of the 13 histidines in carbonmonoxy- and deoxyhemoglobin are known from 1H NMR studies. Those of ACTA are not so precisely known. We employed the Wyman equation and the 13 histidine pairs of pKas to determine the pKas of ACTA by curve-fitting to hemoglobin Bohr Effect data. Using all 14 pairs of pKas as preliminary data, we employed the Wyman equation to fit the Bohr data for hemoglobin chemically modified at Cys93β with cystamine, cystine and iodoacetamide. We demonstrate quantitatively that the reduction of the Bohr Effect upon chemical modification is due to three negatively contributing Bohr groups: His2β, His77β and His143β. These make twice their normal contributions to the Bohr Effect in unmodified hemoglobin. We also find that the ACTA pKas increase with increasing ionic strength.
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Bohr Effect of avian hemoglobins quantitative analyses based on the wyman equation
Journal of Theoretical Biology, 2016Co-Authors: Kehinde O. OkonjoAbstract:The Bohr Effect data for bar-headed goose, greylag goose and pheasant hemoglobins can be fitted with the Wyman equation for the Bohr Effect, but under one proviso: that the pKa of His146β does not change following the T→R quaternary transition. This assumption is based on the x-ray structure of bar-headed goose hemoglobin, which shows that the salt-bridge formed between His146β and Asp94β in human deoxyhemoglobin is not formed in goose deoxyhemoglobin. When the Bohr data for chicken hemoglobin were fitted by making the same assumption, the pKa of the NH3+ terminal group of Val1α decreased from 7.76 to 6.48 following the T→R transition. When the data were fitted without making any assumption, the pKa of the NH3+ terminal group increased from 7.57 to 7.77 following the T→R transition. We demonstrate that avian hemoglobin Bohr data are readily fitted with the Wyman equation because avian hemoglobins lack His77β. From curve-fitting to Bohr data we estimate the pKas of the NH3+ terminal group of Val1α in the R and T states to be 6.33±0.1 and 7.22±0.1, respectively. We provide evidence indicating that these pKas are more accurate than estimates from kinetic studies.
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Bohr Effect of hemoglobins accounting for differences in magnitude
Journal of Theoretical Biology, 2015Co-Authors: Kehinde O. OkonjoAbstract:The basis of the difference in the Bohr Effect of various hemoglobins has remained enigmatic for decades. Fourteen amino acid residues, identical in pairs and located at specific 'Bohr group positions' in human hemoglobin, are implicated in the Bohr Effect. All 14 are present in mouse, 11 in dog, eight in pigeon and 13 in guinea pig hemoglobin. The Bohr data for human and mouse hemoglobin are identical: the 14 Bohr groups appear at identical positions in both molecules. The dog data are different from the human because three Bohr group positions are occupied by non-ionizable groups in dog hemoglobin; the pigeon data are vastly different from the human because six Bohr group positions are occupied by non-ionizable groups in pigeon hemoglobin. The guinea pig data are quite complex. Quantitative analyses showed that only the pigeon data could be fitted with the Wyman equation for the Bohr Effect. We demonstrate that, apart from guinea pig hemoglobin, the difference between the Bohr Effect of each of the other hemoglobins and of pigeon hemoglobin can be accounted for quantitatively on the basis of the occupation of some of their Bohr group positions by non-ionizable groups in pigeon hemoglobin. We attribute the anomalous guinea pig result to a new salt-bridge formed in its R2 quaternary structure between the terminal NH3(+) group of one β-chain and the COO(-) terminal group of the partner β-chain in the same molecule. The pKas of this NH3(+) group are 6.33 in the R2 and 4.59 in the T state.
Antonio V Xavier - One of the best experts on this subject based on the ideXlab platform.
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Replacement of lysine 45 by uncharged residues modulates the redox-Bohr Effect in tetraheme cytochrome c3 of Desulfovibrio vulgaris (Hildenborough).
Biochemistry, 1998Co-Authors: Lígia M. Saraiva, Jean Legall, Carlos A. Salgueiro, P.n. Da Costa, Ana C. Messias, W.m.a.m. Van Dongen, Antonio V XavierAbstract:The structural basis for the pH dependence of the redox potential in the tetrahemic Desulfovibrio vulgaris (Hildenborough) cytochrome c3 was investigated by site-directed mutagenesis of charged residues in the vicinity of heme I. Mutation of lysine 45, located in the neighborhood of the propionates of heme I, by uncharged residues, namely threonine, glutamine and leucine, was performed. The replacement of a conserved charged residue, aspartate 7, present in the N-terminal region and near heme I was also attempted. The analysis of the redox interactions as well as the redox-Bohr behavior of the mutated cytochromes c3 allowed the conclusion that residue 45 has a functional role in the control of the pKa of the propionate groups of heme I and confirms the involvement of this residue in the redox-Bohr Effect.
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redox Bohr Effect in electron proton energy transduction cytochrome c3 coupled to hydrogenase works as a proton thruster in desulfovibrio vulgaris
Journal of Biological Inorganic Chemistry, 1997Co-Authors: Ricardo O. Louro, Teresa Catarino, Jean Legall, Antonio V XavierAbstract:A central step in the metabolism of Desulfovibrio spp. is the oxidation of molecular hydrogen catalyzed by a periplasmic hydrogenase. However, this enzymatic activity is quite low at physiological pH. The hypothesis that, in the presence of the tetrahaem cytochrome c3, hydrogenase can maintain full activity at physiological pH through the concerted capture of the resulting electrons and protons by the cytochrome was tested for the case of Desulfovibrio vulgaris (Hildenborough). The crucial step involves an electron-to-proton energy transduction, and is achieved through a network of cooperativities between redox and ionizable centers within the cytochrome (redox-Bohr Effect). This mechanism, which requires a relocation of the proposed proton channel in the hydrogenase structure, is similar to that proposed for the transmembrane proton pumps, and is the first example which shows evidence of functional energy transduction in the absence of a membrane confinement.
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redox Bohr Effect in electron proton energy transduction cytochrome c 3 coupled to hydrogenase works as a proton thruster in desulfovibrio vulgaris
Journal of Biological Inorganic Chemistry, 1997Co-Authors: Ricardo O. Louro, Teresa Catarino, Jean Legall, Antonio V XavierAbstract:A central step in the metabolism of Desulfovibrio spp. is the oxidation of molecular hydrogen catalyzed by a periplasmic hydrogenase. However, this enzymatic activity is quite low at physiological pH. The hypothesis that, in the presence of the tetrahaem cytochrome c 3, hydrogenase can maintain full activity at physiological pH through the concerted capture of the resulting electrons and protons by the cytochrome was tested for the case of Desulfovibrio vulgaris (Hildenborough). The crucial step involves an electron-to-proton energy transduction, and is achieved through a network of cooperativities between redox and ionizable centers within the cytochrome (redox-Bohr Effect). This mechanism, which requires a relocation of the proposed proton channel in the hydrogenase structure, is similar to that proposed for the transmembrane proton pumps, and is the first example which shows evidence of functional energy transduction in the absence of a membrane confinement.
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Redox-Bohr Effect in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris: a model for energy transduction mechanisms
JBIC Journal of Biological Inorganic Chemistry, 1996Co-Authors: Ricardo O. Louro, Teresa Catarino, Jean Legall, Carlos A. Salgueiro, Antonio V XavierAbstract:Using potentiometric titrations, two protons were found to participate in the redox-Bohr Effect observed for cytochrome c3 from Desulfovibrio vulgaris (Hildenborough). Within the framework of the thermodynamic model previously presented, this finding supports the occurrence of a concerted proton-assisted 2e– step, ideally suited for the coupling role of cytochrome c3 to hydrogenase. Furthermore, at physiological pH, it is shown that when sulfate-reducing bacteria use H2 as energy source, cytochrome c3 can be used as a charge separation device, achieving energy transduction by energising protons which can be left in the acidic periplasmic side and transferring deenergised electrons to sulfate respiration. This mechanism for energy transduction, using a full thermodynamic data set, is compared to that put forward to explain the proton-pumping function of cytochrome c oxidase.
Ming Zou - One of the best experts on this subject based on the ideXlab platform.
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assessment of roles of surface histidyl residues in the molecular basis of the Bohr Effect and of beta 143 histidine in the binding of 2 3 bisphosphoglycerate in human normal adult hemoglobin
Biochemistry, 1999Co-Authors: Tsueiyun Fang, Ming Zou, Virgil SimplaceanuAbstract:Site-directed mutagenesis has been used to construct two mutant recombinant hemoglobins (rHbs), rHb(betaH116Q) and rHb(betaH143S). Purified rHbs were used to assign the C2 proton resonances of beta116His and beta143His and to resolve the ambiguous assignments made over the past years. In the present work, we have identified the C2 proton resonances of two surface histidyl residues of the beta chain, beta116His and beta143His, in both the carbonmonoxy and deoxy forms, by comparing the proton nuclear magnetic resonance (NMR) spectra of human normal adult hemoglobin (Hb A) with those of rHbs. Current assignments plus other previous assignments complete the assignments for all 24 surface histidyl residues of human normal adult hemoglobin. The individual pK values of 24 histidyl residues of Hb A were also measured in deuterium oxide (D(2)O) in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (HEPES) buffer in the presence of 0.1 M chloride at 29 degrees C by monitoring the shifts of the C2 proton resonances of the histidyl residues as a function of pH. Among those surface histidyl residues, beta146His has the biggest contribution to the alkaline Bohr Effect (63% at pH 7.4), and beta143His has the biggest contribution to the acid Bohr Effect (71% at pH 5.1). alpha20His, alpha112His, and beta117His have essentially no contribution; alpha50His, alpha72His, alpha89His, beta97His, and beta116His have moderate positive contributions; and beta2His and beta77His have a moderate negative contribution to the Bohr Effect. The sum of the contributions from 24 surface histidyl residues accounted for 86% of the alkaline Bohr Effect at pH 7.4 and about 55% of the acid Bohr Effect at pH 5.1. Although beta143His is located in the binding site for 2,3-bisphosphoglycerate (2,3-BPG) according to the crystal structure of deoxy-Hb A complexed with 2, 3-BPG, beta143His is not essential for the binding of 2,3-BPG in the neutral pH range according to the proton NMR and oxygen affinity studies presented here. With the accurately measured and assigned individual pK values for all surface histidyl residues, it is now possible to evaluate the Bohr Effect microscopically for novel recombinant Hbs with important functional properties, such as low oxygen affinity and high cooperativity. The present study further confirms the importance of a global electrostatic network in regulating the Bohr Effect of the hemoglobin molecule.
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contribution of surface histidyl residues in the α chain to the Bohr Effect of human normal adult hemoglobin roles of global electrostatic Effects
Biochemistry, 1997Co-Authors: Dazhen Philip Sun, Ming ZouAbstract:We have applied site-directed mutagenesis to our Escherichia coli hemoglobin expression plasmid and constructed five recombinant mutant hemoglobins (r Hbs): r Hb(α20His→Gln or α:H20Q); r Hb(α:H50Q); r Hb(α:H72Q); r Hb(α:H89Q); and r Hb(α:H112Q). We have constructed these r Hbs to help us assess the contribution of the surface histidyl residues in the α-chain to the alkaline Bohr Effect of human normal adult hemoglobin (Hb A). In our laboratory, we have monitored the variation of proton nuclear magnetic resonances arising from the C2 protons of the histidyl residues of Hb A as a function of pH and buffer conditions. Several of these resonances have been assigned to the individual histidyl residues on the surface of the hemoglobin molecule using naturally occurring mutant hemoglobins and chemically modified hemoglobins. In the present work, we have identified the C2 proton resonances of five surface histidyl residues of the α-chain, α20, α50, α72, α89, and α112, in both the carbonmonoxy and deoxy forms, by...
James E Mace - One of the best experts on this subject based on the ideXlab platform.
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roles of the beta 146 histidyl residue in the molecular basis of the Bohr Effect of hemoglobin a proton nuclear magnetic resonance study
Biochemistry, 1991Co-Authors: Mark R Busch, James E MaceAbstract:Assessment of the roles of the carboxyl-terminal beta 146 histidyl residues in the alkaline Bohr Effect in human normal adult hemoglobin by high-resolution proton nuclear magnetic resonance spectroscopy requires assignment of the resonances corresponding to these residues. Previous resonance assignments in low ionic strength buffers for the beta 146 histidyl residue in the carbonmonoxy form of hemoglobin have been controversial [see Ho and Russu (1987) Biochemistry 26, 6299-6305; and references therein]. By a careful spectroscopic study of human normal adult hemoglobin, enzymatically prepared des(His146 beta)-hemoglobin, and the mutant hemoglobins Cowtown (beta 146His----Leu) and York (beta 146His----Pro), we have resolved some of these conflicting results. By a close incremental variation of pH over a wide range in chloride-free 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer, a single resonance has been found to be consistently missing in the proton nuclear magnetic resonance spectra of these hemoglobin variants. The spectra of each of these variants show additional perturbations; therefore, the assignment has been confirmed by an incremental titration of buffer conditions to benchmark conditions, i.e., 0.2 M phosphate, where the assignment of this resonance is unambiguous. The strategy of incremental titration of buffer conditions also allows extension of this resonance assignment to spectra taken in 0.1 M [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane buffer. Participation of the beta 146 histidyl residues in the Bohr Effect has been calculated from the pK values determined for the assigned resonances in chloride-free 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer. Our results indicate that the contribution of the beta 146 histidyl residues is 0.52 H+/hemoglobin tetramer at pH 7.6, markedly less than the 0.8 H+/hemoglobin tetramer estimated by study of the mutant hemoglobin Cowtown (beta 146His----Leu) by Shih and Perutz [(1987) J. Mol. Biol. 195, 419-422]. We have found that at least two histidyl residues in the carbonmonoxy form of this mutant have pK values that are perturbed, and we suggest that these pK differences may in part account for this discrepancy. Furthermore, summation of the positive contribution of the beta 146 histidyl residues and the negative contribution of the beta 2 histidyl residues to the maximum Bohr Effect measured in 0.1 M N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid buffer suggests that additional sites in the hemoglobin molecule account for proton release upon ligation greater than the contribution of the beta 146 histidyl residues.(ABSTRACT TRUNCATED AT 400 WORDS)
Jean Legall - One of the best experts on this subject based on the ideXlab platform.
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Replacement of lysine 45 by uncharged residues modulates the redox-Bohr Effect in tetraheme cytochrome c3 of Desulfovibrio vulgaris (Hildenborough).
Biochemistry, 1998Co-Authors: Lígia M. Saraiva, Jean Legall, Carlos A. Salgueiro, P.n. Da Costa, Ana C. Messias, W.m.a.m. Van Dongen, Antonio V XavierAbstract:The structural basis for the pH dependence of the redox potential in the tetrahemic Desulfovibrio vulgaris (Hildenborough) cytochrome c3 was investigated by site-directed mutagenesis of charged residues in the vicinity of heme I. Mutation of lysine 45, located in the neighborhood of the propionates of heme I, by uncharged residues, namely threonine, glutamine and leucine, was performed. The replacement of a conserved charged residue, aspartate 7, present in the N-terminal region and near heme I was also attempted. The analysis of the redox interactions as well as the redox-Bohr behavior of the mutated cytochromes c3 allowed the conclusion that residue 45 has a functional role in the control of the pKa of the propionate groups of heme I and confirms the involvement of this residue in the redox-Bohr Effect.
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redox Bohr Effect in electron proton energy transduction cytochrome c3 coupled to hydrogenase works as a proton thruster in desulfovibrio vulgaris
Journal of Biological Inorganic Chemistry, 1997Co-Authors: Ricardo O. Louro, Teresa Catarino, Jean Legall, Antonio V XavierAbstract:A central step in the metabolism of Desulfovibrio spp. is the oxidation of molecular hydrogen catalyzed by a periplasmic hydrogenase. However, this enzymatic activity is quite low at physiological pH. The hypothesis that, in the presence of the tetrahaem cytochrome c3, hydrogenase can maintain full activity at physiological pH through the concerted capture of the resulting electrons and protons by the cytochrome was tested for the case of Desulfovibrio vulgaris (Hildenborough). The crucial step involves an electron-to-proton energy transduction, and is achieved through a network of cooperativities between redox and ionizable centers within the cytochrome (redox-Bohr Effect). This mechanism, which requires a relocation of the proposed proton channel in the hydrogenase structure, is similar to that proposed for the transmembrane proton pumps, and is the first example which shows evidence of functional energy transduction in the absence of a membrane confinement.
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redox Bohr Effect in electron proton energy transduction cytochrome c 3 coupled to hydrogenase works as a proton thruster in desulfovibrio vulgaris
Journal of Biological Inorganic Chemistry, 1997Co-Authors: Ricardo O. Louro, Teresa Catarino, Jean Legall, Antonio V XavierAbstract:A central step in the metabolism of Desulfovibrio spp. is the oxidation of molecular hydrogen catalyzed by a periplasmic hydrogenase. However, this enzymatic activity is quite low at physiological pH. The hypothesis that, in the presence of the tetrahaem cytochrome c 3, hydrogenase can maintain full activity at physiological pH through the concerted capture of the resulting electrons and protons by the cytochrome was tested for the case of Desulfovibrio vulgaris (Hildenborough). The crucial step involves an electron-to-proton energy transduction, and is achieved through a network of cooperativities between redox and ionizable centers within the cytochrome (redox-Bohr Effect). This mechanism, which requires a relocation of the proposed proton channel in the hydrogenase structure, is similar to that proposed for the transmembrane proton pumps, and is the first example which shows evidence of functional energy transduction in the absence of a membrane confinement.
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Redox-Bohr Effect in the tetrahaem cytochrome c3 from Desulfovibrio vulgaris: a model for energy transduction mechanisms
JBIC Journal of Biological Inorganic Chemistry, 1996Co-Authors: Ricardo O. Louro, Teresa Catarino, Jean Legall, Carlos A. Salgueiro, Antonio V XavierAbstract:Using potentiometric titrations, two protons were found to participate in the redox-Bohr Effect observed for cytochrome c3 from Desulfovibrio vulgaris (Hildenborough). Within the framework of the thermodynamic model previously presented, this finding supports the occurrence of a concerted proton-assisted 2e– step, ideally suited for the coupling role of cytochrome c3 to hydrogenase. Furthermore, at physiological pH, it is shown that when sulfate-reducing bacteria use H2 as energy source, cytochrome c3 can be used as a charge separation device, achieving energy transduction by energising protons which can be left in the acidic periplasmic side and transferring deenergised electrons to sulfate respiration. This mechanism for energy transduction, using a full thermodynamic data set, is compared to that put forward to explain the proton-pumping function of cytochrome c oxidase.