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The unique Hemoglobin I of L. pectinata: A study of the Heme (FeIV=O) Moiety


The peroxidative reactions of hemoglobin (Hb) and myoglobin (Mb) constrain delivery and turnover of oxygen to cells. The reactions of these proteins with hydrogen peroxide (H2 O2 ) and its superoxide anions are detrimental in red cell pathological conditions, i.e. sickle cell anemia, thalassemia, and red cell aging. These reactions limit the development of proteins for oxygen carrying therapeutics. The formation of ferryl (hemeFe(IV)=O) species have been identified as one of the main reaction intermediates. Interactions of these species with a number of Hb and Mb heme pocket amino acids are also known. Despite the efforts, concerns persist about the transient structure, mechanism, kinetics, dynamics, and reactivity of Hb and Mb with H2 O2 .

The relationship between the structure, reactivity, and function of these Hb and Mb ferryl derivatives is not clearly understood. An answer to these problems will unravel the nature of the ferryl species and their relationship to heme peroxidative reactions.

Our preliminary studies show that we will achieve this goal by taking advantage of the ability of hemoglobin I (HbI) from L. pectinata to stabilize, through its unusual heme pocket configuration (Gln64, Phe29, and Phe68), the ferryl (hemeFe(IV)=O) compound I a thousand times more than Mb. This suggest that the interplay between the proximal trans-ligand effect, the distal heme pocket polarizability, and the absence of hydrogen bonding between the distal amino and the ferryl are also responsible for such behavior.

Here we will address these issues by continuing our previous work on mutated HbI species and following the formation of their heme-ferryl derivative by time resolved resonance Raman. These techniques can detect the transient ferryl species in times shorter than milliseconds and can help resolve questions such as:

  • How does H2 O2 ligand stabilization occur?
  • Which conformational and structural changes are important in the stabilization of HbIFe IV =O ferryl ?
  • What control heme and H2 O2 reactivity in this unique protein?
  • How do the heme pocket aromatic residues contribute or modulate the association and dissociation rate constants for ferryl intermediates in HbI or HbII?


Sebastian Fernandez-Alberti , Daniel E. Bacelo, Robert C. Binning Jr., Julian Echave, Majed Chergui and Juan Lopez-Garriga.
“Sulfide-Binding Hemoglobins: Effects of Mutations on Active-Site Flexibility”.
Biophysical Journal. 2006 Sep 1; 91(5):1698-709. Epub 2006 Jun 16.

Walleska De Jesús-Bonilla, Anthony Cruz, Ariel Lewis, José Cerda, Daniel E. Bacelo, Carmen L. Cadilla and Juan López-Garriga
"Hydrogen-bonding conformations of tyrosine B10 tailor the hemeprotein reactivity of ferryl species"
Journal of Biological Inorganic Chemistry. Volume 11, Number 3 / April, 2006. Pages 334-342.

EJ. A. Gavira, W. de Jesus, A. Camara-Artigas, J. López-Garriga and J. M. García-Ruiz.
"Capillary crystallization and molecular-replacement solution of haemoglobin II from the clam Lucina pectinata". Acta Crystallographica Section F: Structural Biology and Crystallization Communications. (2006). F62, 196-199

Ruth Pietri, Ruth G. León, Laurent Kiger, Michael C. Marden, Laura B. Granell, Carmen L. Cadilla and Juan López-Garriga.
"Hemoglobin I from Lucina pectinata: A model for distal heme-ligand control".
Biochim et Biophys Acta. 2006 Apr;1764(4):758-65. Epub 2005 Dec 12.

Pietri R, Granell L, Cruz A, De Jesus W, Lewis A, Leon R, Cadilla CL, Garriga JL..
"Tyrosine B10 and heme-ligand interactions of Lucina pectinata hemoglobin II: control of heme reactivity".
Biochim Biophys Acta. 2005 Mar 14;1747(2):195-203. Epub 2004 Dec 19.

Ruth Gretchen León, Hélène Munier-Lehmann, Octavian Barzu, Véronique Baudin-Creuza, Ruth Pietri, Juan López-Garriga and Carmen L. Cadilla.
"High-level production of recombinant sulfide-reactive hemoglobin I from Lucina pectinata in Escherichia coli. High yields of fully functional holoprotein synthesis in the BLi5 E. coli strain".
Protein Expression and Purification Volume 38, Issue 2 , December 2004, Pages 184-195.

Jan Helbing , Luigi Bonacina, Ruth Pietri, Jens Bredenbeck, Peter Hamm, Frank van Mourik , Frédéric Chaussard , Alejandro Gonzalez-Gonzalez , Majed Chergui, Cacimar Ramos-Alvarez , Carlos Ruiz and Juan López-Garriga.
"Time-Resolved Visible and Infrared Study of the Cyano Complexes of Myoglobin and of Hemoglobin I from Lucina pectinata".
Biophysical Journal 87:1881-1891 (2004).

E. Ramirez, C. Ramos, M. Rodríguez, R. Pietri, M. Chergui and J. Lopez-Garriga
"Ultrafast studies of ferric complexes of Hemoglobin I from Lucina pectinata Femtochemistry and Femtobiology".
(Ed. M.M Martin, J.T. Hynes) Elsevier N.Y. pages 395-398.

Evys Collazo, Ruth Pietri, Walleska De Jesús, Cacimar Ramos, Ana Del Toro, Ruth Gretchen León, Carmen L. Cadilla and Juan López-Garriga.
"Functional Characterization of the Purified Holo Form of Hemoglobin I from Lucina pectinata Overexpressed in Escherichia coli".
The Protein Journal. Volume 23, Number 4 ,May 2004, Pages: 239 - 245.

Elineth Torres-Mercado, Jessicca Y. Renta, Yolanda Rodríguez, Juan López-Garriga and Carmen L. Cadilla.
"The cDNA-Derived Amino Acid Sequence of Hemoglobin II from Lucina pectinata".
Journal of Protein Chemistry 2003 Nov;22(7-8):683-90.

Torres-Mercado,E., Lopez-Garriga,J. and Cadilla-Vazquez,C.L.
The cDNA Derived Amino Acid Sequence of Hemoglobin II from Lucina pectinata.
Genbank, Accesion AY243364. http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nucleotide&val=29692327.

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