Abstract
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.
Highlights
Introduction ofGln64(E7) into the wild-type MbCO crystal structure based on its orientation in the H64Q-MbCO crystal structure results in reasonable agreement with both T1 data and ␦dip(calc)
Gln64(E7) in the L29F/H64Q/V68F-metMbH2O crystal structure leads to an excellent fit for ␦dip(obs) versus ␦dip(calc) and yields RFe(N⑀H) ϭ 4.2 Å, in good agreement with the 4.2-Å value obtained from the T1 ϭ 14 ms via Equation 5
Small variations in 1, 2 starting with the Gln64(E7) orientation in the single mutant lead to a Gln orientation essentially the same as that obtained starting with the coordinate of the triple mutant
Summary
Protein Preparation—The pUC19 plasmid containing V68F sperm whale myoglobin was made by Egeberg et al [31]. Experimental dipolar shifts for the structurally conserved proximal side of the heme were used as input to search for the Euler rotation angles, ⌫(␣,,␥), that transforms the molecular pseudo-symmetry coordinates (xЈ, yЈ, zЈ, or R, Ј, ⍀Ј (Fig. 1)) readily obtained from crystal coordinates [17, 24, 25, 51] into magnetic axes, x, y, and z, by minimizing the global error function,. Minimizing the error function F/n in Equation 1 was performed over three parameters, ␣, , and ␥, using available ⌬ax and ⌬rh, or extended to all five parameters to yield both the Euler angles and anisotropies as described in detail previously [29]. Dipolar Shift Simulations—The position of a substituted or perturbed residue can be determined by minimizing a local error function. The molecular modeling was carried out on a Silicon Graphics INDIGO from available crystal coordinates [17, 24, 25, 51] using the INSIGHT II (Biosym/MSI, San Diego) and MIDAS (UCSF) programs
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