The Apolar Channel in Cerebratulus lacteus Hemoglobin Is the Route for O2 Entry and Exit

Salter, M.D.; Nienhaus, Karin; Nienhaus, G. Ulrich; Dewilde, Sylvia; Moëns, Luc; Pesce, Alessandra; Nardini, M.; Bolognesi, Martino; Olson, John S.

doi:10.1074/jbc.m805727200

Cited by 31 publications

(61 citation statements)

References 57 publications

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“…, which is close to the diffusion limit (32,38,51,61 , effectively blocking the E7 channel, and there is a roughly monotonic decrease in the rate of NO binding with increasing size of the E7 side chain for other mutants. Scott et al (17) carried out a much more extensive mutagenesis mapping study of Mb using 90 mutants at 27 different positions, and their results confirmed quantitatively the E7 gate mechanism for ligand entry into Mb, which was originally developed using just E7 mutations (60).…”

Section: E7 Gate Appears To Be the Pathway For Ligand Entry Into Hba-mentioning

confidence: 99%

“…Kinetic Measurements of O 2 , CO, and NO Binding-Parameters for O 2 binding to the Hb E7 mutant tetramers and monomers were measured using laser flash photolysis of the stable HbCO complex in O 2 /CO mixtures to avoid prolonged exposure to oxygen and autoxidation (32). Descriptions of the 0.5-s dye laser system and sample preparations are given in supplemental Section B.…”

Section: Methodsmentioning

confidence: 99%

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Distal Histidine Stabilizes Bound O2 and Acts as a Gate for Ligand Entry in Both Subunits of Adult Human Hemoglobin

Birukou

Schweers²,

Olson

2010

Journal of Biological Chemistry

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174

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The role of the distal histidine in regulating ligand binding to adult human hemoglobin (HbA) was re-examined systematically by preparing His(E7) to Gly, Ala, Leu, Gln, Phe, and Trp mutants of both Hb subunits. Rate constants for O 2 , CO, and NO binding were measured using rapid mixing and laser photolysis experiments designed to minimize autoxidation of the unstable apolar E7 mutants. Replacing His(E7) with Gly, Ala, Leu, or Phe causes 20 -500-fold increases in the rates of O 2 dissociation from either Hb subunit, demonstrating unambiguously that the native His(E7) imidazole side chain forms a strong hydrogen bond with bound O 2 in both the ␣ and ␤ chains (⌬G His(E7)H-bond ≈ ؊8 kJ/mol). As the size of the E7 amino acid is increased from Gly to Phe, decreases in k O2 , In 1970, Perutz (1) proposed that the distal histidines located at the E7 helical positions, 3 ␣His-58 and ␤His-63, play crucial structural roles for regulating both the affinities and rates of O 2 binding to adult human hemoglobin (HbA). 4 These ideas were based on the suggestion by Pauling (2) that His(E7) could stabilize bound O 2 by donating a hydrogen bond to the partial negative charge on the superoxide-Fe(III)-like FeO 2 complex and on the idea by Perutz and Mathews (3) that the distal histidine could also be acting as gate for ligand entry and exit. Studies of model heme compounds and naturally occurring globins with His(E7) replacements suggested strongly that the distal histidine also plays a key role in discrimination between O 2 and CO binding (4 -9).The first mutagenesis studies on sperm whale Mb and human HbA reported that His(E7) to Gly mutations in ␣ subunits and Mb cause marked increases in the rates of O 2 dissociation and significant decreases in affinity, both of which indicate strong stabilization of the FeO 2 complex by proton donation from the wild-type His(E7) side chain (7, 10, 11). In addition, the association rate constants for O 2 and CO binding increased 5-10-fold. The latter results were interpreted in terms of the distal histidine gate mechanism, with the His(E7) to Gly mutation resulting in an open E7 channel. In contrast, neither the dissociation nor association rate constants for O 2 binding to the R state ␤Gly(E7) mutant of HbA appeared to increase significantly, implying no electrostatic stabilization of bound ligands by the native His(E7) in ␤ subunits and an already open gate or alternative pathway. These surprising kinetic results were explained by the first high resolution structure of human oxyhemoglobin published by Shaanan (12), in which the N⑀H atoms of distal histidine in ␤ subunits seemed to be further away from the bound O 2 atoms and pointing toward the heme plane.Between 1989 and 1999, our group and others constructed large libraries of mammalian Mb mutants as a model system for understanding the structural mechanisms of ligand binding to vertebrate globins involved in O 2 transport and storage. A detailed molecular mechanism for O 2 binding has emerged. Ligand entry into myoglobin occurs through t...

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Section: E7 Gate Appears To Be the Pathway For Ligand Entry Into Hba-mentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Distal Histidine Stabilizes Bound O2 and Acts as a Gate for Ligand Entry in Both Subunits of Adult Human Hemoglobin

Birukou

Schweers²,

Olson

2010

Journal of Biological Chemistry

Self Cite

100

174

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“…CO rebinding to the unliganded intermediates transiently formed after ligand photodissociation was followed after a 0.5-s excitation pulse from a Phase-R model 2100B dye laser, as described in previous work (35,46,47).…”

Section: Methodsmentioning

confidence: 99%

Blocking the Gate to Ligand Entry in Human Hemoglobin

Birukou

Soman

Olson

2011

Journal of Biological Chemistry

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His(E7) to Trp replacements inHemoglobins represent a very diverse protein family with members occurring in all six kingdoms of life (1). The functions of these proteins differ significantly, ranging from oxygen storage and transport, NO dioxygenation, and nitrite reduction to sensing intracellular levels of diatomic gases for transcriptional regulation and chemotaxis (2). The central chemical events for these functions are movement of ligands into the distal portion of the heme pocket, internal bond formation with the heme iron, and electrostatic stabilization or steric hindrance of the bound ligand by surrounding active site amino acids. Although the quantum mechanical details of bond formation remain to be resolved, there is general agreement on the biophysical mechanisms governing steric hindrance and hydrogen bonding between amino acid side chains and the bound ligand (3-10). In contrast, the pathways for ligands movement from solvent through the protein and into the active site are still under debate.As recently reviewed by Elber (11), molecular dynamics simulations and other computational approaches have almost uniformly suggested that there are multiple routes for ligand entry into and escape from the active sites of hemoglobins and myoglobins (12-27). Many of these pathways are coincident with apolar cavities, which have been identified as xenon docking sites (16, 17, 28 -30). However, as also pointed out by Elber (11), there is little or no direct experimental evidence in support of multiple pathways (31, 32). In the cases of mammalian myoglobins, human hemoglobin, and HbI from Scapharca inaequivalvis, almost all of the experimental evidence suggests that Ն75% of ligands enter and exit the distal pocket through a transient channel between the heme propionates, which is produced by outward rotation of the distal histidine (His(E7)). 2Although ligands do migrate into internal cavities immediately after photodissociation, they appear to return to the distal pocket and escape through the E7 gate (33-41).The structures of the active sites and the E7 channels in Mb and the subunits of human HbA are very similar, and thus analogous mechanisms for ligand binding are inferred. Until very recently, there were little experimental and computational data regarding the pathways of ligand migration into HbA. Mouawad et al. (15) observed the formation of transient cavities in the ␣ and ␤ subunits of human HbA during simulations of the T to R conformational change and postulated these cavities could allow ligands to diffuse through the globin matrix. Sottini et al. (16,17) used molecular modeling approaches to find potential xenon binding cavities in human HbA and suggested that ligands could use these apolar voids as pathways to enter or escape the active site. Savino et al. (30) reported crystal structures of Tyr(B10)/Gln(E7) deoxyHbA with xenon atoms partially occupying the sites identified in molecular modeling experiments. Thus, if only the theoretical and structural litera-* This work was supported, in whole or...

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“…Alternate topological features such as interior gas tunnels in truncated hemoglobins, for example, have been shown to be mechanistically important for modulating ligand migration (18,19). However, further studies are needed to fully understand how these related protein frameworks within the globin family contribute to variations in ligand-binding properties.…”

mentioning

confidence: 99%

“…Recent structural studies conducted on noncanonical globins have greatly expanded our knowledge of how globins function (18)(19)(20)(21)(22)(23). Alternate topological features such as interior gas tunnels in truncated hemoglobins, for example, have been shown to be mechanistically important for modulating ligand migration (18,19).…”

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confidence: 99%

Tunnels modulate ligand flux in a heme nitric oxide/oxygen binding (H-NOX) domain

Winter

Herzik

Kuriyan

et al. 2011

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

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Interior topological features, such as pockets and channels, have evolved in proteins to regulate biological functions by facilitating the diffusion of biomolecules. Decades of research using the globins as model heme proteins have clearly highlighted the importance of gas pockets around the heme in controlling the capture and release of O 2 . However, much less is known about how ligand migration contributes to the diverse functions of other heme protein scaffolds. Heme nitric oxide/oxygen binding (H-NOX) domains are a conserved family of gas-sensing heme proteins with a divergent fold that are critical to numerous signaling pathways. Utilizing X-ray crystallography with xenon, a tunnel network has been shown to serve as a molecular pathway for ligand diffusion. Structure-guided mutagenesis results show that the tunnels have unexpected effects on gas-sensing properties in H-NOX domains. The findings provide insights on how the flux of biomolecules through protein scaffolds modulates protein chemistry.

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The Apolar Channel in Cerebratulus lacteus Hemoglobin Is the Route for O2 Entry and Exit

Cited by 31 publications

References 57 publications

Distal Histidine Stabilizes Bound O2 and Acts as a Gate for Ligand Entry in Both Subunits of Adult Human Hemoglobin

Distal Histidine Stabilizes Bound O2 and Acts as a Gate for Ligand Entry in Both Subunits of Adult Human Hemoglobin

Blocking the Gate to Ligand Entry in Human Hemoglobin

Tunnels modulate ligand flux in a heme nitric oxide/oxygen binding (H-NOX) domain

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