Solution of 1H NMR Structure of the Heme Cavity in the Oxygen-avid Myoglobin from the Trematode Paramphistomum epiclitum

Wei Zhang,Khwaja A Rashid,Masoodul Haque,Ather H Siddiqi,Serge N Vinogradov,Luc Moens,Gerd N La Mar

doi:10.1074/jbc.272.5.3000

Abstract

A two-dimensional 1H NMR study has been carried out on the heme cavity of the extreme oxygen-avid and autoxidation-resistant oxy-myoglobin complex from the trematode Paramphistomum epiclitum, and the residues were identified which potentially provide hydrogen bond stabilization for the bound oxygen. Complete assignment of the heme core resonances allows the identification of 10 key heme pocket residues, 4 Phe, 4 Tyr, and 2 upfield ring current aliphatic side chains. Based solely on the conserved myoglobin folding topology that places the E helix-heme crossover and the completely conserved Phe(CD1)-heme contact at opposing meso positions, the heme orientation in the cavity and the E helix alignment were unambiguously established that place Tyr66 at position E7. Moreover, all eight aromatic and the two aliphatic side chains were shown to occupy the positions in the heme cavity predicted by amino acid sequence alignment with globins of known tertiary structure. The dipolar contacts for the Tyr32(B10) and Tyr66(E7) rings indicate that both residues are oriented into the heme cavity, which is unprecedented in globins. The ring hydroxyl protons for both Tyr are close to each other and in a position to provide hydrogen bonds to the coordinated oxygen, as supported by strong retardation of their exchange rate with bulk solvent. A more crowded and compact structure increases the dynamic stability of the distal pocket and may contribute to the autoxidation resistance of this myoglobin.

Highlights

Myoglobin and hemoglobin are oxygen binding proteins that reflect extraordinary structural homology in spite of often very limited sequence homology [1, 2]
Hbs and Mbs from invertebrates exhibit a much broader range of functionality and show key substitutions at position E7 that are unknown among vertebrates and provide hydrogen bonds to the bound O2 via residues at positions other than E7 [2]
The Tyr(B10) hydrogen bond has been identified as the source of the extra stabilization that leads to extreme O2 avidity in A. suum HbI through primarily an extremely slow O2 off-rate [9, 12]; the autoxidizability is similar to that of common Mbs/Hbs

Summary

The abbreviations used are

Hemoglobin; Mb, myoglobin; NOESY, two-dimensional nuclear Overhauser spectroscopy; TOCSY, two-dimensional total correlation spectroscopy; DSS, 2,2Ј-dimethyl-2pentane-5-sulfonate. Hbs and Mbs from invertebrates exhibit a much broader range of functionality and show key substitutions at position E7 that are unknown among vertebrates and provide hydrogen bonds to the bound O2 via residues at positions other than E7 [2] Prominent among these globins are the Mb from the sea hare Aplysia limacina, which, with a Val(E7), provides the key hydrogen bond via Arg(E10) [8], and the parasitic trematode, Ascaris suum, HbI, which provides two hydrogen bonds to the bound O2 via a relatively common Gln(E7) and an uncommon Tyr(B10) [9, 10]. Pure, with the remainder one or more of the minor Mb isoforms This sample heterogeneity limits the scope of the study but not the details to which the positions of the identified heme cavity residues can be placed relative to each other and to the heme

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION