Ligand-Linked Changes at the Subunit Interfaces in Scapharca Hemoglobins Probed through the Sulfhydryl Infrared Absorption

Abstract

FTIR spectra of native Scapharca homodimeric hemoglobin (HbI) and of the Phe97-->Ile mutant have been measured in the region 2400-2700 cm(-1) where the absorption of the sulfhydryl groups can be observed. In native HbI, the two Cys92 residues give rise to a relatively intense band centered at 2559 cm(-1) that is shifted to 2568 cm(-1) and strongly quenched upon ligand binding. In the Phe97-->Leu mutant, such ligand-linked changes are not observed and the strong peak at around 2560 cm(-1) persists in the liganded derivatives. In native HbI, the observed changes have been attributed to the decrease in polarity of the interface due to the ligand-induced extrusion of the Phe97 phenyl ring from the heme pocket to the interface and the subsequent release of several water molecules that are clustered in the vicinity of Cys92. In contrast, in the Phe97-->Leu mutant, the Leu residue does not leave the heme pocket upon ligand binding and the interface is unaltered. The Cys92/S-H infrared band, therefore, represents a sensitive probe of the structural rearrangements that take place in the intersubunit interface upon ligand binding to HbI. The heterotetrameric Scapharca hemoglobin HbII contains, in addition to the Cys92 residues in the interfaces, two extra sulfhydryl groups per tetramer (Cys9 in the B chain) that are exposed to solvent in the A helix. The frequency of the Cys9/S-H stretching vibration occurs at 2582 cm(-1) in the unliganded and at 2586 cm(-1) in the liganded derivative, pointing to the involvement of the A helix in the ligand-linked polymerization characteristic of HbII.

Insight into protein structure and protein-ligand recognition by Fourier transform infrared spectroscopy

“…In addition, hydrogen bonding from the tyrosine to other groups leads to a shift of this band. The S-H sulfhydryl group of cystein absorbs between 2400 and 2700 cm À1 in H 2 O and around 1857 cm À1 in D 2 O (Tu, 1986;Gregoriou et al, 1995;Guarrera et al, 1999). S-H groups which are hydrogen bonded show significantly lower S-H stretch modes down to 2290 cm À1 (Sellmann et al, 1991;Boorman et al, 1992).…”

Protein Structural Dynamics of Wild-Type and Mutant Homodimeric Hemoglobin Studied by Time-Resolved X-Ray Solution Scattering

“…However, the allosteric structural transition of Hb is still elusive because of the complex kinetics arising from its heteromeric tetramer structure. In this respect, homodimeric hemoglobin (HbI) from Scapharca inaequivalvis has served as an excellent model system for investigating the allosteric structural transition between a ligated R state with high ligand affinity and a deoxygenated T state with low ligand affinity [ 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 ] due to its simpler dimeric structure. In the HbI, the E and F helices of monomers are located at the interface of homodimer [ 11 , 12 , 39 ], which is often referred to as EF dimer.…”