Role of heme in the unfolding and assembly of myoglobin.

The unfolding of wild-type holomyoglobin in the ferric state (metMb) appears to be a simple two-state process, even though hemichrome spectra are often observed and apoMb denaturation involves an intermediate. To resolve these discrepancies, we measured GuHCl-induced, equilibrium unfolding of five sperm whale metMb variants, which were selected to examine the relative importance of apoglobin stability and hemin affinity. Combined analysis of CD, Trp fluorescence, and Soret absorbance titration curves for all the variants requires a six-state mechanism containing native (N), intermediate (I), and unfolded (U) states of apoMb and their hemin-bound counterparts, NH (holoMb), IH, and UH, respectively.