Heme pocket disorder in myoglobin: Reversal by acid-induced soft refolding

The protein folding process of heme proteins entails generation of not only a correct global polypeptide structure, but also a correct, functionally c ompetent heme environment. We employed a variety of spectroscopic approache s to probe the structure and dynamics of the heme pocket of a recombinant s perm whale myoglobin. The conformational characteristics were examined by c ircular dichroism, time-resolved fluorescence spectroscopy, FTIR spectrosco py, and optical absorption spectroscopy in the temperature range 300-20 K. Each of these spectroscopic probes detected modifications confined exclusiv ely to the heme pocket of the expressed myoglobin relative to the native pr otein. The functional properties were examined by measuring the kinetics of CO binding after flash-photolysis. The kinetics of the expressed myoglobin were more heterogeneous than those of the native protein. Mild acid exposu re of the ferric derivative of the recombinant protein resulted in a protei n with "nativelike" spectroscopic properties and homogeneous CO binding kin etics. The heme pocket modifications observed in this recombinant myoglobin do not derive from inverted heme. In contrast, when native apomyoglobin is reconstituted with the heme in vitro, the heme pocket disorder could be at tributed exclusively to 180 degrees 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 affin ity of globin for the heme and allows an extended conformational sampling o r "soft refolding" to a nativelike conformation.