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J Biol Chem, Vol. 275, Issue 2, 742-751, January 14, 2000

Solution ¹H NMR Study of the Influence of Distal Hydrogen Bonding and N Terminus Acetylation on the Active Site Electronic and Molecular Structure of Aplysia limacina Cyanomet Myoglobin^*

Bao D. Nguyen, Zhicheng Xia, Francesca Cutruzzolá^§, Carlo Travaglini Allocatelli^§, Maurizio Brunori^§, and Gerd N. La Mar^¶

From the  Department of Chemistry, University of California, Davis, California 95616 and the ^§ Dipartimento di Scienze Biochimiche, University of Rome "La Sapienza," "A. Rossi Fanelli" P. le A. Moro, 5, I-00185 Rome, Italy

The sea hare Aplysia limacina possesses a myoglobin in which a distal H-bond is provided by Arg E10 rather than the common His E7. Solution ¹H NMR studies of the cyanomet complexes of true wild-type (WT), recombinant wild-type (rWT), and the V(E7)H/R(E10)T and V(E7)H mutants of Aplysia Mb designed to mimic the mammalian Mb heme pocket reveal that the distal His in the mutants is rotated out of the heme pocket and is unable to provide a stabilizing H-bond to bound ligand and that WT and rWT differ both in the thermodynamics of heme orientational disorder and in heme contact shift pattern. The mean of the four heme methyl shifts is shown to serve as a sensitive indicator of variations in distal H-bonding among a set of mutant cyanomet globins. The heme pocket perturbations in rWT relative to WT were traced to the absence of the N-terminal acetyl group in rWT that participates in an H-bond to the EF corner in WT. Analysis of dipolar contacts between heme and axial His and between heme and the protein matrix reveal a small ~2° rotation of the axial His in rWT relative to true WT and a ~3° rotation of the heme in the double mutant relative to rWT Mb. It is demonstrated that both the direction and magnitude of the rotation of the axial His relative to the heme can be determined from the change in the pattern of the contact-dominated heme methyl shift and from the dipolar-dominated heme meso-H shift. However, only NOE data can determine whether it is the His or heme that actually rotates in the protein matrix.

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				Z. Xia, B. D. Nguyen, M. Brunori, F. Cutruzzola, and G. N. La Mar 1H-NMR Study of the Effect of Temperature through Reversible Unfolding on the Heme Pocket Molecular Structure and Magnetic Properties of Aplysia limacina Cyano-Metmyoglobin Biophys. J., December 1, 2005; 89(6): 4149 - 4158. [Abstract] [Full Text] [PDF]