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
Zhicheng Xia 1, Bao D Nguyen 2, Maurizio Brunori 3*, Francesca Cutruzzola' 3 and Gerd N La Mar 4
1 Department of Chemistry, McGill University, Montreal, Quebec, Canada
2 Department of Chemistry, University of California, Irvine, California,USA
3 Dipartimento Scienze Biochimiche, Università Roma La Sapienza, Rome, Italy
4 Department of Chemistry, University California Davis USA
* To whom correspondence should be addressed. E-mail: maurizio.brunori{at}uniroma1.it.
Submitted on April 6, 2005
Revised on June 1, 2005
Accepted on 27 July 2005
 |
Abstract |
|---|
2D 1H NMR spectroscopy over a range of temperature through thermal unfolding has been applied to the low-spin, ferric cyanide complex of myoglobin from Aplysia limacina to search for intermediates in the unfolding and to characterize the effect of temperature on the magnetic properties and electronic structure of the heme iron. The observation of strictly linear behavior from 5 to 80 °C through the unfolding transition for all hyperfine shifted resonances indicates the absence of significant populations of intermediate states to the cooperative unfolding with Tm ~ 80°C. The magnetic anisotropies and orientation of the magnetic axes for the complete range of temperatures were also determined for the complex. The anisotropies have very similar magnitudes, and exhibit the expected characteristic temperature dependence, previously observed in the isoelectronic sperm whale myoglobin complex. In contrast to sperm whale Mb, where the orientation of the magnetic axis was completely temperature-independent, the tilt of the major magnetic axis, which correlates with the Fe-CN tilt, decrease at high temperature in Aplysia limacina Mb, indicating a molecular structure that is conserved with temperature, although more plastic than that of sperm whale Mb. The pattern of contact shifts are shown to reflect a conserved Fe-His(F8) bond and 
spin delocalization into the heme as expected for the orientation of the axial His imidazole.
Key Words:
NMR, dipolar shifts, magnetic anisotropy, magnetic axes, myoglobin, thermal unfolding
