| Abstract
| - The protein folding process of heme proteins entails generation of not only a correct globalpolypeptide structure, but also a correct, functionally competent heme environment. We employed a variety ofspectroscopic approaches to probe the structure and dynamics of the heme pocket of a recombinant spermwhale myoglobin. The conformational characteristics were examined by circular dichroism, time-resolvedfluorescence spectroscopy, FTIR spectroscopy, and optical absorption spectroscopy in the temperature range300−20 K. Each of these spectroscopic probes detected modifications confined exclusively to the heme pocketof the expressed myoglobin relative to the native protein. The functional properties were examined by measuringthe kinetics of CO binding after flash-photolysis. The kinetics of the expressed myoglobin were moreheterogeneous than those of the native protein. Mild acid exposure of the ferric derivative of the recombinantprotein resulted in a protein with “nativelike” spectroscopic properties and homogeneous CO binding kinetics.The heme pocket modifications observed in this recombinant myoglobin do not derive from inverted heme. Incontrast, when native apomyoglobin is reconstituted with the heme in vitro, the heme pocket disorder could beattributed exclusively to 180° rotation of the bound heme [La Mar, G. N., Toi, H., and Krishnamoorthi, R.(1984) J. Am. Chem. Soc. 106, 6395−6401; Light, W. R., Rohlfs, R. J., Palmer, G., and Olson, J. S. (1987) J.Biol. Chem. 262, 46−52]. We conclude that exposure to low pH decreases the affinity of globin for the hemeand allows an extended conformational sampling or “soft refolding” to a nativelike conformation.
|
