Structure and Ligand Selection of Hemoglobin II from Lucina pectinata

Gavira, José A.; Cámara-Artigas, A.; Jesús-Bonilla, Walleska De; López‐Garriga, Juan; Lewis, Ariel; Pietri, Ruth; Yeh, Syun Ru; Cadilla, Carmen L.; García‐Ruiz, Juan Manuel

doi:10.1074/jbc.m705026200

Cited by 24 publications

(39 citation statements)

References 52 publications

(56 reference statements)

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“…Indeed, as Fig. 1 shows, the presence of these residues was recently confirmed by the X-ray crystal structure of HbII [9]. The structure of HbII also reveals that oxygen is tightly bound to the heme through hydrogen bonds with TyrB10 and GlnE7, and that the heme group is buried farther than in HbI.…”

Section: Introductionmentioning

confidence: 63%

“…Figure 6b shows that titration of the purified rHbII with potassium ferricyanide did not induced full oxidation of the protein. In fact, similar to nHbII [9], to oxidize the rHbII heme center a decrease in pH from 7.5 to 5 was required. Taken together, these results suggest that both, nHbII and rHbII have similar heme electronic and distal structural properties.…”

Section: Resultsmentioning

confidence: 99%

“…In addition, the fact that to fully oxidize rHbII required a decrease in pH, indicates that, as in nHbII, the recombinant protein is resistant to oxidation of the iron heme moiety due to the architecture of the heme distal environment. Regarding this, previous results have demonstrated that the orientation of TyrB10, reduces the heme cavity of HbII and the HbI PheB10Tyr mutant and that this reduction may help to prevent oxidation of the ferrous iron center by impeding the access of external water molecules into the distal environment [9]. Hence, this finding along with the slow auto-oxidation kinetics observed here strongly suggests that architecture of the rHbII heme distal environment is similar to that of the nHbII and that mutations of the rHbII distal residues can provide relevant information about ligand discrimination in Lucina pectinata as well as other organisms.…”

Section: Discussionmentioning

confidence: 99%

“…The structure of HbII also reveals that oxygen is tightly bound to the heme through hydrogen bonds with TyrB10 and GlnE7, and that the heme group is buried farther than in HbI. These findings led to the suggestion that the combined effects of the hydrogen bonding interactions involving TyrB10 and GlnE7, and the localization of the heme pocket, confer stability to the oxy-HbII complex and prevent oxidation of the ferrous iron center [9]. This explains in part the capability of HbII to selectively bind oxygen and at the same time, its inability to coordinated H 2 S. This ligand discrimination process facilitates the relationship between the mollusk and its endosymbiont.…”

Section: Introductionmentioning

confidence: 99%

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Recombinant Hemoglobin II From Lucina pectinata: A Large-Scale Method For Hemeprotein Expression in E. coli

et al. 2010

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Hemoglobin II from the clam L. pectinata is an O 2 reactive protein that remains oxygenated in the presence of other molecules. To determine the mechanism of ligand selection in this hemoglobin, rHbII was expressed in large quantities using an improved fermentation process. The highest protein yield was obtained by: transforming HbII into the BLi5 cells, inducing and supplementing the culture during the mid-log phase with 1 mM IPTG, 30 µg/mL hemin chloride and 1% glucose, and decreasing the temperature to 30 °C after induction. In addition, cell culture density was greatly enhanced by using glycerol, adding MgSO 4 , supplementing the media with glucose after the glycerol was consumed and maintaining the dissolved oxygen at 35%. Under these conditions the maximum protein yield obtained was ~2,300 mg/L. The results indicate that rHbII is similar to the native protein.The protocol was validated with other hemoglobins, indicating that it can be extended to other hemeproteins.

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Section: Introductionmentioning

confidence: 63%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Recombinant Hemoglobin II From Lucina pectinata: A Large-Scale Method For Hemeprotein Expression in E. coli

et al. 2010

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“…Structural solutions and refinements of the Lucina crystal structures were carried out as described in earlier publications [18]. …”

Section: Methodsmentioning

confidence: 99%

Extreme differences between Hemoglobins I and II of the Clam Lucina pectinalis in their reactions with nitrite

Bonaventura

Henkens

Jesús-Bonilla

et al. 2010

Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics

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The clam Lucina pectinalis supports its symbiotic bacteria by H 2 S transport in the open and accessible heme pocket of Lucina Hb I and by O 2 transport in the narrow and crowded heme pocket of Lucina Hb II. Remarkably, air-equilibrated samples of Lucina Hb I were found to be more rapidly oxidized by nitrite than any previously studied Hb, while those of Lucina Hb II showed an unprecedented resistance to oxidation induced by nitrite. Nitrite-induced oxidation of Lucina Hb II was enabled only when O 2 was removed from its active site. Structural analysis revealed that O 2 clams up the active site by hydrogen bond formation to B10Tyr and other distal-side residues. Quaternary effects further restrict nitrite entry into the active site and stabilize the hydrogen-bonding network in oxygenated Lucina Hb II dimers. The dramatic differences in nitrite reactivities of the Lucina Hbs are not related to their O 2 affinities or anaerobic redox potentials, which were found to be similar, but are instead a result of differences in accessibility of nitrite to their active sites; i.e. these differences are due to a kinetic rather than thermodynamic effect. Comparative studies revealed heme accessibility to be a factor in human Hb oxidation by nitrite as well, as evidenced by variations of rates of nitrite-induced oxidation that do not correlate with R and T state differences and inhibition of oxidation rate in the presence of O 2 . These results provide a dramatic illustration of how evolution of active sites with varied heme accessibility can moderate the rates of inner-sphere oxidative reactions of Hb and other heme proteins. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBiochim Biophys Acta. Author manuscript; available in PMC 2011 October 1. Published in final edited form as:Biochim Biophys Acta. 2010 October ; 1804(10): 1988-1995. doi:10.1016/j.bbapap.2010.016. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript 1. INTRODUCTIONNitrite is a unique Hb oxidant because of its ability to react with oxyHb in a complex reaction leading to formation of metHb and nitrate [1][2][3][4][5], and with deoxyHb in a reaction that generates metHb and NO [6][7]. Many recent studies have focused on the formation of bioactive NO from nitrite that is catalyzed by deoxy Hb because of the potential, but still controversial, role of this reaction in blood pressure regulation [8][9][10][11][12][13][14].Work in our laboratories on the reactions of normal and cross-linked forms of Hb with nitrite led to some puzzling results. Conditions that ...

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SAXS structure of homodimeric oxyHemoglobin III from bivalve Lucina pectinata

et al. 2021

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Hemoglobin III (HbIII) is one of the two oxygen reactive hemoproteins present in the bivalve, Lucina pectinata. The clam inhabits a sulfur‐rich environment and HbIII is the only hemoprotein present in the system which does not yet have a structure described elsewhere. It is known that HbIII exists as a heterodimer with hemoglobin II (HbII) to generate the stable Oxy(HbII‐HbIII) complex but it remains unknown if HbIII can form a homodimeric species. Here, a new chromatographic methodology to separate OxyHbIII from the HbII‐HbIII dimer has been developed, employing a fast performance liquid chromatography and ionic exchange chromatography column. The nature of OxyHbIII in solution at concentrations from 1.6 mg/mL to 20.4 mg/mL was studied using small angle X‐ray scattering (SAXS). The results show that at all concentrations, the Oxy(HbIII‐HbIII) dimer dominates in solution. However, as the concentration increases to nonphysiological values, 20.4 mg/mL, HbIII forms a 30% tetrameric fraction. Thus, there is a direct relationship between the Oxy(HbIII‐HbIII) oligomeric form and hemoglobin concentration. We suggest it is likely that the OxyHbIII dimer contributes to active oxygen transport in tissues of L pectinata, where the Oxy(HbII‐HbIII) complex is not present.

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Structure and Ligand Selection of Hemoglobin II from Lucina pectinata

Cited by 24 publications

References 52 publications

Recombinant Hemoglobin II From Lucina pectinata: A Large-Scale Method For Hemeprotein Expression in E. coli

Recombinant Hemoglobin II From Lucina pectinata: A Large-Scale Method For Hemeprotein Expression in E. coli

Extreme differences between Hemoglobins I and II of the Clam Lucina pectinalis in their reactions with nitrite

SAXS structure of homodimeric oxyHemoglobin III from bivalve Lucina pectinata

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