Residue F4 Plays a Key Role in Modulating Oxygen Affinity and Cooperativity in <i>Scapharca</i> Dimeric Hemoglobin

Knapp, James E.; Bonham, Michele A.; Gibson, Quentin H.; Nichols, Jeffry C.; Royer, William E.

doi:10.1021/bi051052+

Cited by 29 publications

(64 citation statements)

References 31 publications

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“…Binding of oxygen to crystals of HbI is fully cooperative (20), while associated structural transitions are relatively localized and compatible with a well ordered crystal lattice (21). Despite limited structural differences, the two allosteric states show dramatic functional changes with an Ϸ300-fold difference in oxygen affinity between the high-affinity R-state and the low-affinity T-state (22,23). Three important tertiary structural rearrangements contribute to the differences in oxygen affinity between the two states: movement of Phe-97 (F4) from the subunit interface into the proximal heme pocket (23,24), a redistribution of interfacial water molecules (22), and movement of the heme groups toward the subunit interface (25) (see Movie 1, which is published as supporting information on the PNAS web site).…”

Section: Allosteric Protein Transitions ͉ Intersubunit Communication mentioning

confidence: 99%

Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin

Knapp

Pahl²,

Šrajer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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111

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Protein allostery provides mechanisms for regulation of biological function at the molecular level. We present here an investigation of global, ligand-induced allosteric transition in a protein by time-resolved x-ray diffraction. The study provides a view of structural changes in single crystals of Scapharca dimeric hemoglobin as they proceed in real time, from 5 ns to 80 s after ligand photodissociation. A tertiary intermediate structure forms rapidly (<5 ns) as the protein responds to the presence of an unliganded heme within each R-state protein subunit, with key structural changes observed in the heme groups, neighboring residues, and interface water molecules. This intermediate lays a foundation for the concerted tertiary and quaternary structural changes that occur on a microsecond time scale and are associated with the transition to a low-affinity T-state structure. Reversal of these changes shows a considerable lag as a T-like structure persists well after ligand rebinding, suggesting a slow T-to-R transition.allosteric protein transitions ͉ intersubunit communication ͉ kinetics A fundamental goal in biology is to understand how organisms respond to environmental signals. Central to this process are allosteric proteins that undergo structural transitions between alternate states in response to stimuli such as ligand binding. Although static structures of alternate states are available for a number of allosteric proteins, information about the kinetic pathway between such functionally important states is limited. Time-resolved crystallographic analysis (1-9) provides the unique opportunity to obtain direct, time-dependent structural information at high resolution on the entire protein molecule as it undergoes structural change.Much of our understanding of allosteric protein function has come from investigations into mammalian hemoglobins. Evidence of substantial structural changes accompanying ligand binding dates back to the 1930s with the observation that unliganded, high-salt, hemoglobin crystals exposed to oxygen shatter (10), foreshadowing the discovery of large quaternary changes that are linked to oxygen binding (11). Unliganded crystals of human hemoglobin grown under certain low-salt conditions can maintain their integrity upon oxygen binding; however, oxygen binding in this case is noncooperative (12). Although the ability to follow allosteric transitions in the crystalline state is limited when such large quaternary structural changes accompany cooperative ligand binding, spectroscopic investigations of hemoglobin solutions have revealed some aspects of the kinetic pathway of allosteric structural transitions (13-15). Results to date suggest a multistep pathway, with key tertiary structural transitions taking place within a few microseconds and quaternary rearrangements occurring in the range of tens of microseconds. In addition to providing structural information on high-affinity R and low-affinity T quaternary forms at the atomic level (16-18), crystallographic experiments have also succeeded...

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Section: Allosteric Protein Transitions ͉ Intersubunit Communication mentioning

confidence: 99%

Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin

Knapp

Pahl²,

Šrajer

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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111

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“…F97Y, in particular, has been studied in detail recently (8). The characteristic movement of residue F97, packing close to the heme in the deoxy form and moving out on ligand binding, does not occur.…”

Section: Nature and Assignment Of Signalmentioning

confidence: 99%

“…by site-directed mutagenesis (7,8), and the role of interfacial water probed by the isosteric substitution T72V (9,10). It does not alter the structure apart from these water molecules but the mutation raises oxygen affinity some 50-fold.…”

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confidence: 99%

An Optical Signal Correlated with the Allosteric Transition in Scapharca inaequivalvis HbI

2006

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The transient absorbance change in the first 2 μs after photolysis of COHbI (from Scapharca inaequivalvis) reported by Chiancone et al.(1) has been studied in several mutants. Evidence is presented that this change (rts) is associated with the allosteric transition between R-and T-states. Two different rts spectra relate to Hb 2 and Hb 2 CO. No rts has been observed for mutants at position 97 (normally Phe). Correlation of ligand binding and rts shows that protein function changes at or near the rates of rts -typically 2 × 10 6 s −1 (Hb 2 ) and 5 × 10 5 s −1 (Hb 2 CO). Unique values of allosteric parameters for several mutants have been obtained by combining kinetic and equilibrium data. The effect of mutation on function thus may be assigned to allostery or to change in intrinsic heme reactivity.Cooperativity in ligand binding by mammalian hemoglobins, expressed in their sigmoid equilibrium curves, was established about a century ago. These studies initiated attempts, still ongoing, to describe the curves in physicochemical terms. Difficulties in studying these hemoglobins have arisen from: (a) two kinds of subunits, (b) four subunits per molecule, (c) a strong dependence on pH and salts and (d) tetramer to dimer dissociation. Clearly, a dimeric hemoglobin would be much easier to study. There are, however, few dimeric types of hemoglobin, and fewer still that bind ligands cooperatively. The hemoglobin from Scapharca inaequivalvis that has two identical subunits, no effect of pH and salts, marked cooperativity, and a very small dimer to monomer dissociation constant even when liganded, perhaps comes closest to the ideal. This homodimeric hemoglobin was first studied in relation to an Adriatic clam fishery. References to the early work may be found in Antonini et al. (2). In that paper the kinetics of the reactions of the native protein with oxygen and carbon monoxide were described in terms of the two-state model (MWC) of Monod, Wyman and Changeux (3). They noted, however, that unique values of the allosteric parameters could not be assigned from ligand binding studies alone because of the close correlation between L (the ratio of the R and T forms of deoxyhemoglobin) and c (the change in L on binding each ligand molecule). The structures of both liganded and deoxy forms of the wild type and of many mutants have been determined to high resolution (4,5). Unlike mammalian hemoglobins, there are no major quaternary structural changes on ligand binding, allowing fully cooperative oxygen binding to crystals (6). However, residue F97 does move away from the heme group and water molecules in the interface between the subunits change in number and position. The importance of residue 97 has been underscored † This work was supported by the National Institutes of Health (grants DK43323 and GM66756 to WER)

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“…A total of 10,000 to 100,000 transients were collected with a 100-kHz spectral width, 8 k complex data points, and 600-ms recycling time. 15 N chemical shifts were verified with a solution of 4 M ammonium nitrate in 2 M nitric acid. Only chemical shift differences are reported in the text.…”

Section: Nmr Spectroscopymentioning

confidence: 99%

“…In pentacoordinate globins, substitutions at positions other than the proximal histidine (labeled F8 in the Perutz nomenclature), for example at F4 and F7, generally do not change the coordination and spin state of the iron or the fold of the protein. These residues, however, are often involved in packing interactions (14,15) and hydrogen-bonding networks (16) that adjust essential characteristics of the protein, such as proximal Fe-N bond length and heme reactivity (17,18). Proximal residues control solvent access (19) and contribute to the positioning of the proximal histidine and the iron relative to the heme plane, which, in turn, relate to the affinity for exogenous ligands (20) and the affinity for the heme group (21).…”

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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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Residue F4 Plays a Key Role in Modulating Oxygen Affinity and Cooperativity in Scapharca Dimeric Hemoglobin

Cited by 29 publications

References 31 publications

Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin

Allosteric action in real time: Time-resolved crystallographic studies of a cooperative dimeric hemoglobin

An Optical Signal Correlated with the Allosteric Transition in Scapharca inaequivalvis HbI

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

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