The role of cavities in protein dynamics: Crystal structure of a photolytic intermediate of a mutant myoglobin

We determined the structure of the photolytic intermediate of a sperm whale myoglobin (Mb) mutant called Mb−YQR [Leu−(B10)right−arrowTyr; His(E7)right−arrowGln; Thr(E10)right−arrowArg] to 1.4−Å resolution by ultra−low temperature (20 K) x−ray diffraction. Starting with the CO complex, illumination leads to photolysis of the Fe−CO bond, and migration of the photolyzed carbon monoxide (CO*) to a niche in the protein 8.1 ? from the heme iron; this cavity corresponds to that hosting an atom of Xe when the crystal is equilibrated with xenon gas at 7 atmospheres [Tilton, R. F., Jr., Kuntz, I. D. & Petsko, G. A. (1984) Biochemistry 23, 2849−2857]. The site occupied by CO* corresponds to that predicted by molecular dynamics simulations previously carried out to account for the NO geminate rebinding of Mb−YQR observed in laser photolysis experiments at room temperature. This secondary docking site differs from the primary docking site identified by previous crystallographic studies on the photolyzed intermediate of wild−type sperm whale Mb performed at cryogenic temperatures [Teng et al. (1994) Nat. Struct. Biol. 1, 701−705] and room temperature [Šrajer et al. (1996) Science 274, 1726−1729]. Our experiment shows that the pathway of a small molecule in its trajectory through a protein may be modified by site−directed mutagenesis, and that migration within the protein matrix to the active site involves a limited number of pre−existing cavities identified in the interior space of the protei