An order-disorder transition plays a role in switching off the root effect in fish hemoglobins.
The Root effect is a widespread property among fish hemoglobins whose structural basis remains largely obscure. Here we report a crystallographic and spectroscopic characterization of the non-Root-effect hemoglobin isolated from the Antarctic fish Trematomus newnesi in the deoxygenated form. The crystal structure unveils that the T state of this hemoglobin is stabilized by a strong H-bond between the side chains of Asp95α and Asp101β at the α(1)β(2) and α(2)β(1) interfaces. This unexpected finding undermines the accepted paradigm that correlates the presence of this unusual H-bond with the occurrence of the Root effect. Surprisingly, the T state is characterized by an atypical flexibility of two α chains within the tetramer. Indeed, regions such as the CDα corner and the EFα pocket, which are normally well ordered in the T state of tetrameric hemoglobins, display high B-factors and non-continuous electron densities. This flexibility also leads to unusual distances between the heme iron and the proximal and distal His residues. These observations are in line with Raman micro-spectroscopy studies carried out both in solution and in the crystal state. The findings here presented suggest that in fish hemoglobins the Root effect may be switched off through a significant destabilization of the T state regardless of the presence of the inter-aspartic H-bond. Similar mechanisms may also operate for other non-Root effect hemoglobins. The implications of the flexibility of the CDα corner for the mechanism of the T-R transition in tetrameric hemoglobins are also discussed.
Vergara
Alessandro
A
Department of Chemistry, University of Naples Federico II, Naples I-80126, Italy.
Vitagliano
Luigi
L
Merlino
Antonello
A
Sica
Filomena
F
Marino
Katia
K
Verde
Cinzia
C
di Prisco
Guido
G
Mazzarella
Lelio
L
D000818:Animals; D018360:Crystallography, X-Ray; D005399:Fishes; D006454:Hemoglobins / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D013059:Spectrum Analysis, Raman
D006454:Hemoglobins