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

Abstract

The O 2 -avid hemoglobin from the parasitic nematode Ascaris suum exhibits one of the slowest known O 2 off rates. Solution 1 H NMR has been used to investigate the electronic and molecular structural properties of the active site for the cyano-met derivative of the recombinant first domain of this protein. Assignment of the heme, axial His, and majority of the residues in contact with the heme reveals a molecular structure that is the same as reported in the A. suum HbO 2 crystal structure (Yang, J., Kloek, A., Goldberg, D. E., and Mathews, F. S. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 4224 -4228) with the exception that the heme in solution is rotated by 180°about the ␣,␥-meso axis relative to that in the crystal. The observed dipolar shifts, together with the crystal coordinates of HbO 2 , provide the orientation of the magnetic axes in the molecular framework. The major magnetic axis, which correlates with the Fe-CN vector, is found oriented ϳ30°away from the heme normal and indicates significant steric tilt because of interaction with Tyr 30 (B10). The three side chain labile protons for the distal residues Tyr 30 (B10) and Gln 64 (E7) were identified, and their relaxation, dipolar shifts, and nuclear Overhauser effects to adjacent residues used to place them in the distal pocket. It is shown that these two distal residues exhibit the same orientations ideal for H bonding to the ligand and to each other, as found in the A. suum HbO 2 crystal. It is concluded that the ligated cyanide participates in the same distal H bonding network as ligated O 2 . The combination of the strong steric tilt of the bound cyanide and slow ring reorientation of the Tyr 30 (B10) side chain supports a crowded and constrained distal pocket.The O 2 binding globins, myoglobin (Mb) 1 and hemoglobin (Hb), despite highly varied sequences throughout phylogeny, possess a highly conserved folding topology of 7-8 helices (A-H), with the heme wedged in between the E and F helices and ligated by one, His F8 (proximal), of only two (the other is Phe CD1) completely conserved residues (1-3). Despite this strong structural homology, the O 2 ligation rates and O 2 affinities vary over a remarkably wide range (by ϳ10 5 ), depending on the exact nature of several distal residues at the key positions B10, E7, and E10 (4 -8). The most important distal interaction for stabilizing bound O 2 is hydrogen bonding to the ligand, for which the donor is generally His E7 (1, 7, 9, 10) and is Gln E7 in a few cases (2). In several invertebrates, such as Aplysia and Dolbella Mbs that possess a Val E7, the distal H bond to the ligand is provided by an Arg at position E10 (11-13).A particularly noteworthy class of globins is that of parasitic nematodes that possess, in addition to a H bond donor at position E7, a Tyr at position B10 that is also capable of H bonding to the ligand (4, 5, 8, 14 -17 (5,15). The positions of these two key residues are clearly defined in the crystal structure of A. suum HbO 2 , in which the two residues are appropriately poised ...

Controlling Ligand Binding in Myoglobin by Mutagenesis

“…(36,47). Similar evidence for steric crowding was observed in the structure of A. suum cyanometHb (48).…”

Globin-like proteins in Caenorhabditis elegans: in vivo localization, ligand binding and structural properties

“…This Hb has an exceptionally high O 2 affinity (K O2 = k on / k off : 0.215 M -1 ; P 50 : 0.001-0.004 torr at 20°C) that is attributed to a very slow dissociation rate ( k off (O 2 ): 0.0041 s -1 ) and a normal association rate ( k on (O 2 ): 1.5 μM -1 s -1 ) [6], whereby this Hb will be in the oxy form even in the host's gut with a locally micro-oxygen concentration [6]. This high O 2 affinity can be explained structurally by the presence of three hydrogen bonds between TyrB10 and GlnE7 and the bound ligand [7,8]. The function of the A. suum pseudocoelomic fluid Hb is still a matter of debate (for a review see: [5,6]).…”

1H NMR study of the molecular structure and magnetic properties of the active site for the cyanomet complex of O2‐avid hemoglobin from the trematode Paramphistomum epiclitum

“…For the present Pe metHbCN, the Tyr32(B10) C ε Hs signal exhibits <10 Hz excess linewidth at 15 °C that could result from the role of ring reorientation. The magnetic axes yield a chemical shift difference of 6.6 p.p.m., very similar to that in Ascaris metHbCN [37]. Hence the narrow Tyr32(B10) C ε H signal dictates that the rate of Tyr32(B10) ring reorientation is >10 2 times faster in Pe metHbCN than As metHbCN.…”

“…It has been proposed that the pattern of the meso‐H hyperfine shift is determined largely by dipolar shifts due to the rhombic anisotropy [30,36,37]. The Δ(obs) = 1/2[δ hf (5‐H) − δ hf (10‐H) + δ hf (15‐H) − δ hf (20‐H)] = 8.2 p.p.m., while Δ(calc) = 1/2[δdip(5‐H) − δdip(10‐H) + δdip(15‐H) − δdip(20‐H)] = 9 ± 2 p.p.m., which confirms the prediction.…”

“…Two such rings of interest are Tyr32(B10) and Phe46(CD1). The TyrB10 ring of the nematode Ascaris metHbCN with relatively ‘normal’ autoxidation rate exhibits [37]≈ 450 Hz excess line broadening at 15 °C that could be attributed to slow reorientation of the ring. The difference in the TyrB10 C ε H chemical shifts, as determined from the magnetic axes [37], was found to be 6.0 p.p.m.…”