Sequential assignment of proton resonances in the NMR spectrum of Zn-substituted α chains from human hemoglobin.

We constructed an artificial holoprotein as a complex between α globin from human adult hemoglobin and the protoporphyrin IX-Zn(II).The prosthetic group is bound in a single conformation to the apoglobin via a coordinative bond between Zn(II) ion and the proximal histidine (His87).The complex is diamagnetic and does not bind either CO nor O₂ thus representing a diamagnetic model of deoxygenated α chains.In the present paper we report extensive resonance assignment in the proton nuclear magnetic resonance spectrum of the Zn-substituted α chains in phospate buffer pH 5.6.A large number of aromatic and aliphatic side chain spin systems were identified in the two-dimensional homonuclear COSY spectra.Based on the assigned resonance of heme substituent protons and their NOE cross-peaks, we assigned the majority of resonances representing the heme pocket ide chains. Using the main-chain-directed assignment strategy, we could establish several continuous patterns of sequential assignment and identify partial or total spin systems for a large number of side chains.The final assignment corresponds to 73% of the amino acids. Analysis of chemical shift of assigned resonances and of nuclear Overhauser enhancement connectivities provides structural information on the global and local tertiary conformation in solution and on the ligand-induced conformational changes. Comparison of observed and calculated ring current shifts enabled us to compare the solution structure with the X-ray crystal structure of α subunits in deoxy and carbonmonoxy hemoglobin. The global tertiary structure of unliganded chains is highly similar to both liganded and unliganded counterparts in the crystalline state. On the distal side of the heme pocket, Va162 is significantly closer to the heme center, in agreement with its conformation in the crystallographic structure.In contrast, the position of the proximal histidine (His87) relative to the heme is clearly more closely related to that in the liganded tetramer in the crystalline state. Comparison of the chemical shift values for the resonances in carbon monoxy and Zn(II)-substituted α chains indicates that the ligand-induced conformational changes are essentially localized in the heme pocket area and effect proximal side residues more than the distal side ones. Some notable for the functional mechanism. [ABSTRACT FROM AUTHOR]