Thr-E11 regulates O2 affinity in Cerebratulus lacteus mini-hemoglobin

Alessandra Pesce, Marco Nardini, Paolo Ascenzi, Eva Geuens, Sylvia Dewilde, Luc Moens, Martino Bolognesi, Austen F. Riggs, Angela Hale, Pengchi Deng, G. Ulrich Nienhaus, John S. Olson, Karin Nienhaus

Research output: Contribution to journalArticle

Abstract

The mini-hemoglobin from Cerebratulus lacteus (CerHb) belongs to a class of globins containing the polar Tyr-B10/Gln-E7 amino acid pair that normally causes low rates of O2 dissociation and ultra-high O2 affinity, which suggest O2 sensing or NO scavenging functions. CerHb, however, has high rates of O2 dissociation (kO2 = 200-600 s-1) and moderate O2 affinity (KO2 ≈1 μM-1) as a result of a third polar amino acid in its active site, Thr-E11. When Thr-E11 is replaced by Val, kO2 decreases 1000-fold and KO2 increases 130-fold at pH 7.0, 20 °C. The mutation also shifts the stretching frequencies of both heme-bound and photodissociated CO, indicating marked changes of the electrostatic field at the active site. The crystal structure of Thr-E11 → Val CerHbO2 at 1.70 Å resolution is almost identical to that of the wild-type protein (root mean square deviation of 0.12 Å). The dramatic functional and spectral effects of the Thr-E11 → Val mutation are due exclusively to changes in the hydrogen bonding network in the active site. Replacing Thr-E11 with Val "frees" the Tyr-B10 hydroxyl group to rotate toward and donate a strong hydrogen bond to the heme-bound ligand, causing a selective increase in O2 affinity, a decrease of the rate coefficient for O2 dissociation, a 40 cm-1 decrease in VCO of heme-bound CO, and an increase in ligand migration toward more remote intermediate sites.

Original languageEnglish
Pages (from-to)33662-33672
Number of pages11
JournalJournal of Biological Chemistry
Volume279
Issue number32
DOIs
Publication statusPublished - Aug 6 2004

ASJC Scopus subject areas

  • Biochemistry

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    Pesce, A., Nardini, M., Ascenzi, P., Geuens, E., Dewilde, S., Moens, L., Bolognesi, M., Riggs, A. F., Hale, A., Deng, P., Nienhaus, G. U., Olson, J. S., & Nienhaus, K. (2004). Thr-E11 regulates O2 affinity in Cerebratulus lacteus mini-hemoglobin. Journal of Biological Chemistry, 279(32), 33662-33672. https://doi.org/10.1074/jbc.M403597200