SOLUTION AND CRYSTAL-STRUCTURES OF A SPERM WHALE MYOGLOBIN TRIPLE MUTANT THAT MIMICS THE SULFIDE-BINDING HEMOGLOBIN FROM LUCINA PECTINATA

The bivalve mollusc Lucina pectinata harbors sulfide-oxidizing chemoau totrophic bacteria and expresses a monomeric hemoglobin I, HbI, with n ormal O-2, but extraordinarily high sulfide affinity. The crystal stru cture of aquomet Lucina HbI has revealed an active site with three res idues 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 mu tant with similar to 700-fold higher sulfide affinity than for wild-ty pe. The single crystal x-ray structure of the aquomet derivative of th e myoglobin triple mutant and the solution H-1 NMR active site structu res of the cyanomet derivatives of both the myoglobin mutant and Lucin a 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 g roup acts as H-bond donor to the bound ligand. Phe(68)(E11) is rotated similar to 90 degrees about chi(2) and located similar to 1-2 Angstro m closer to the iron atom in the myoglobin triple mutant relative to i ts conformation in Lucina HbI. The change in orientation potentially e liminates the stabilizing interaction with sulfide and, together with the decrease in size of the distal pocket, accounts for the 7-fold low er sulfide affinity of the myoglobin mutant compared with that of Luci na HbI.