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 chemoautotrophic bacteria and expresses a monomeric hemoglobin I, HbI, with normal O2, 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 approximately 700-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 1H 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 Gln64(E7) serves as a H-bond acceptor, whereas in the cyanomet form the amido group acts as H-bond donor to the bound ligand. Phe68(E11) is rotated approximately 90 degrees about chi2 and located approximately 1-2 A 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.