Solution and Crystal Structures of a Sperm Whale Myoglobin Triple Mutant That Mimics the Sulfide-binding Hemoglobin fromLucina pectinata

Abstract

The bivalve mollusc Lucina pectinata harbors sulfideoxidizing chemoautotrophic bacteria and expresses a monomeric hemoglobin I, HbI, with normal O 2 , but extraordinarily high sulfide affinity. The crystal structure of aquomet Lucina HbI has revealed an active site with three residues not commonly found in vertebrate globins: Phe(B10), Gln(E7), and Phe(E11) (Rizzi, M., Wittenberg, J. B., Coda, A., Fasano, M., Ascenzi, P., and Bolognesi, M. (1994) J. Mol. Biol. 244, 86 -89). Engineering these three residues into sperm whale myoglobin results in a triple mutant with ϳ700-fold higher sulfide affinity than for wild-type. The single crystal x-ray structure of the aquomet derivative of the myoglobin triple mutant and the solution 1 H NMR active site structures of the cyanomet derivatives of both the myoglobin mutant and Lucina HbI have been determined to examine further the structural origin of their unusually high sulfide affinities. The major differences in the distal pocket is that in the aquomet form the carbonyl of Gln 64 (E7) serves as a H-bond acceptor, whereas in the cyanomet form the amido group acts as H-bond donor to the bound ligand. Phe 68 (E11) is rotated ϳ90°about 2 and located ϳ1-2 Å closer to the iron atom in the myoglobin triple mutant relative to its conformation in Lucina HbI. The change in orientation potentially eliminates the stabilizing interaction with sulfide and, together with the decrease in size of the distal pocket, accounts for the 7-fold lower sulfide affinity of the myoglobin mutant compared with that of Lucina HbI.

1H NMR Investigation of the Distal Hydrogen Bonding Network and Ligand Tilt in the Cyanomet Complex of Oxygen-avidAscaris suum Hemoglobin

“…4. The magnitude of the tilt of the major magnetic axis, z, from the heme normal (zЈ axis), ␤ ϭ ϳ30°, is nearly twice as much as that observed previously in cyanomet globins (34,50,51). The large magnitude of the tilt of the major magnetic axis from the heme normal indicated by the complete magnetic axes determination also reveals itself clearly in the analysis of the dipolar shift pattern for individual residues.…”

“…A comparison can be made to globins with Phe rather than Tyr(B10) and with a Gln(E7), i.e. elephant Mb and the sperm whale Leu 29 (B10) 3 Phe/ His 64 (E7) 3 Gln and Leu 29 (B10) 3 Phe/His 64 (E7) 3 Gln/ Val 68 (E11) 3 Phe Mb mutants, for which the B10 ring exhibited "normal" linewidth indicative of much faster reorientation (39,51). Whether the constraints on the Tyr 30 (B10) ring in A. suum Hb result from "pinning down" the extremity via the H bond to the ligand or from the tight van der Waals' contacts with the aromatic ring is not known but could be elucidated in a comparison of the solution 1 H NMR spectra of WT and Tyr(B10) 3 Phe A. suum mutant Hb.…”

“…Magnetic Axes-The magnetic axes 2 were determined as described in detail previously (34,50,51). Experimental dipolar shifts, ␦ DSS (obs), for protons on nonligated residues for structurally conserved portions (relative to A. suum HbO 2 ) of the heme environment were used as input to search for the Euler rotation angles, ⌫ (␣, ␤, ␥), that transforms the iron-centered pseudo-symmetry coordinates (xЈ, yЈ, zЈ, or R, Ј, ⍀Ј) (Fig.…”

“…Magnetic Axes-The orientation of the magnetic axes 2 was found to be essentially independent of the selection of input data or whether the anisotropies were also determined or held constant at the values determined for sperm whale metMbCN (50,51). The resulting orientation of is defined by ␤ ϭ 29.5°Ϯ…”

Mapping hydration dynamics around a protein surface

“…(v) The distinct water residence times are selected for different mutated locations from recent MD simulations for all 297 hydration sites (42). (vi) Finally, the structural stability of other mutants from various previous studies (43)(44)(45).…”