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J. Biol. Chem., Vol. 279, Issue 32, 33662-33672, August 6, 2004

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Thr-E11 Regulates O₂ Affinity in Cerebratulus lacteus Mini-hemoglobin^*

From the ^aDepartment of Physics-INFM and Center for Excellence in Biomedical Research, University of Genova, Via Dodecaneso 33, 16146 Genova, Italy, the ^bDepartment of Biology and Interdepartmental Laboratory of Electron Microscopy, University "Roma Tre," Viale G. Marconi 446, 00146 Roma, Italy, the ^cDepartment of Biomedical Sciences, University of Antwerp, Universiteitsplein 1, 2610 Antwerp, Belgium, ^eStructural Biology Unit, National Institute for Cancer Research, Largo R. Benzi 10, 16132 Genova, Italy, ^gSection of Neurobiology, University of Texas, Austin, Texas 78712-0252, the ⁱDepartment of Biochemistry and Cell Biology and the W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005-1892, the ^jDepartment of Biophysics, University of Ulm, Albert-Einstein-Allee 11, 89081 Ulm, Germany, and the ^kDepartment of Physics, University of Illinois, Urbana, Illinois 61801-3080

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 (k_O2 = 200-600 s^-1) and moderate O₂ affinity (K_O2 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, k_O2 decreases 1000-fold and K_O2 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 _CO of heme-bound CO, and an increase in ligand migration toward more remote intermediate sites.

Received for publication, April 1, 2004 , and in revised form, May 21, 2004.

^* This work was supported in part by grants from the Italian National Research Council (Functional Genomics Project), from the FIRB Project Grant RBAU015B47_002, "Protein Folding," and from the European Union Project "Neuroglobin and Survival of the Neuron" Grant QLG3-CT-2002-01548 (to M. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^d Postdoctoral fellow of the Fund for Scientific Research Flanders (FWO).

^f Supported by the Fondazione Compagnia di S. Paolo (Turin, Italy) and the Istituto G. Gaslini (Genova, Italy).

^h Supported by National Science Foundation Grant MCB-0237651.

^l Supported by Deutsche Forschungsgemeinschaft Grant Ni291/3 and the "Fonds der Chemischen Industrie."

^m Supported by United States Public Health Service Grants GM 35649 and HL 47020 from the National Institutes of Health and Robert A. Welch Foundation Grant C-612.

ⁿ To whom correspondence should be addressed. Tel.: 49-731-5023049; Fax: 49-731-5023059; E-mail: Karin.Nienhaus{at}physik.uni-ulm.de.

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