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. The unfolding parameters for the apoMbs were obtained in independent experiments and then fixed in the analysis of the holoprotein data, where only the affinities of the apoglobin states for hemin were allowed to vary. This cofactor binding analysis applies generally to all globins and led to three specific conclusions. (1) The stability of holo-metMb is determined primarily by the high affinity (K(d) approximately 10(-13) M) of native apoMb (N) for hemin. (2) The partially unfolded intermediate with hemin bound (IH) has a hemichrome spectrum indicative of a bis-histidyl axial coordination and is seen clearly when the stability of the N state or its affinity for hemin is reduced. (3) Although the affinity of the intermediate for hemin (K(d) approximately 10(-11) M) is approximately 100-fold lower than that for the native state, free hemin can bind to it and promote the assembly of the holoprotein.
Culbertson
David S
DS
Department of Biochemistry and Cell Biology and W. M. Keck Center for Computational Biology, Rice University, Houston, Texas 77005, USA.
Olson
John S
JS
D000818:Animals; D005453:Fluorescence; D006418:Heme / Q000737:chemistry*; D006427:Hemin / Q000737:chemistry; D008786:Metmyoglobin / Q000737:chemistry* / Q000235:genetics; D017510:Protein Folding; D017433:Protein Structure, Secondary; D014907:Whales
D008786:Metmyoglobin; D006418:Heme; D006427:Hemin