DESIGN CHALLENGES FOR HEMOPROTEINS - THE SOLUTION STRUCTURE OF APOCYTOCHROME B(5)

In order to characterize the structural and dynamic factors that deter mine the assembly in b hemoproteins, the solution structure of the 98- residue protein apocytochrome bs was determined by NMR methods. Over 8 00 experimental restraints derived from a series of two- and three-dim ensional experiments were used. Holocytochrome b(5), the protein with iron protoporphyrin-IX liganded to His-39 and His-63, contains in sequ ence the following elements of secondary structure: beta 1-alpha-1-bet a 4-beta 3-alpha 2-alpha 3-beta 5-alpha 4-alpha 5-beta 2-alpha 6 [Math ews, F. S., Czerwinski, E. W., & Argos, P. (1979) The Porphyrins, Vol. 7, pp 107-147, Academic Press, New York]. The folded holoprotein poss esses two hydrophobic cores: an extensive, functional core around the heme (core 1), and a smaller, structural core remote from the heme (co re 2). The apoprotein was found to contain a stable four-stranded beta -sheet encompassing beta 1, beta 2, beta 3, and beta 4 and three alpha -helices, corresponding to alpha 1, alpha 2, and alpha 6. Two short al pha-helices (alpha 3 and alpha 5) appear to form partially, and alpha 4 is not detected. These three helices and beta 5 border the heme bind ing pocket and are disordered in the apoprotein NMR structure. Accordi ng to backbone H-1-N-15 NOE results, the most flexible region of the a poprotein, except for the termini, extends from Ala-50 (in beta 5) to Glu-69 (in alpha 5). The polypeptide segment bearing His-63 (located i mmediately prior to alpha 5) exhibits faster internal motions than tha t bearing His-39 (at the C-terminal end of alpha 2). The latter imidaz ole samples a restricted region of space, whereas the former can adopt many orientations with respect to the stable core. It was concluded t hat heme removal affects the structure and dynamics of most of core 1 whereas it leaves core 2 largely intact. The results provide guideline s for the rational design of b hemoproteins: a modular structure inclu ding a packed, stable core and a partially folded binding site is anti cipated to present strong kinetic and thermodynamic advantages compare d to approaches relying on the complete formation of secondary structu re prior to heme binding.