Chlamydomonas chloroplast ferrous hemoglobin: Heme pocket structure and reactions with ligands

Manon Couture, Tapan Kanti Das, H. Caroline Lee, Jack Peisach, Denis L. Rousseau, Beatrice A. Wittenberg, Jonathan B. Wittenberg, Michel Guertin

Research output: Contribution to journalArticle

108 Scopus citations

Abstract

We report the optical and resonance Raman spectral characterization of ferrous recombinant Chlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6- coordinate low spin species at alkaline pH. The proximal ligand to the heme is the imidazole group of a histidine. Kinetics of the reactions with ligands were determined by stopped-flow spectroscopy. At alkaline pH, combination with oxygen, nitric oxide, and carbon monoxide displays a kinetic behavior that is interpreted as being rate-limited by conversion of the 6-coordinate form to a reactive 5-coordinate form. At neutral pH, combination rates of the 5-coordinate form with oxygen and carbon monoxide were much faster (>107 μM-1 s-1). The dissociation rate constant measured for oxygen is among the slowest known, 0.014 s-1, and is independent of pH. Replacement of the tyrosine 63 (B10) by leucine or of the putative distal glutamine by glycine increases the dissociation rate constant 70- and 30-fold and increases the rate of autoxidation 20- and 90-fold, respectively. These results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the distal glutamine. In addition, the high frequency (232 cm-1) of the iron-histidine bond suggests a structure that lacks any proximal strain thus contributing to high ligand affinity.

Original languageEnglish (US)
Pages (from-to)6898-6910
Number of pages13
JournalJournal of Biological Chemistry
Volume274
Issue number11
DOIs
StatePublished - Mar 12 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Couture, M., Das, T. K., Lee, H. C., Peisach, J., Rousseau, D. L., Wittenberg, B. A., Wittenberg, J. B., & Guertin, M. (1999). Chlamydomonas chloroplast ferrous hemoglobin: Heme pocket structure and reactions with ligands. Journal of Biological Chemistry, 274(11), 6898-6910. https://doi.org/10.1074/jbc.274.11.6898