Structural Insights into the Heme Pocket and Oligomeric State of Non-Symbiotic Hemoglobins from Arabidopsis thaliana.

Alessandra Astegno,Mariarita Bertoldi,Carolina Conter,Paola Dominici

doi:10.3390/biom10121615

Abstract

Non-symbiotic hemoglobins AHb1 and AHb2 from Arabidopsis thaliana are hexacoordinate heme-proteins that likely have different biological roles, in view of diverse tissue localization, expression pattern, and ligand binding properties. Herein, we expand upon previous biophysical studies on these isoforms, focusing on their oligomeric states and circular dichroism (CD) characteristics. We found that AHb1 exists in solution in a concentration-dependent monomer-dimer equilibrium, while AHb2 is present only as a monomer. The quaternary structure of AHb1 affects its degree of hexacoordination with the formation of the dimer that enhances pentacoordination. Accordingly, the mutant of a conserved residue within the dimeric interface, AHb1-T45A, which is mostly monomeric in solution, has an equilibrium that is shifted toward a hexacoordinate form compared to the wild-type protein. CD studies further support differences in the globin’s structure and heme moiety. The Soret CD spectra for AHb2 are opposite in sense to those for AHb1, reflecting different patterns of heme-protein side chain contacts in the two proteins. Moreover, the smaller contribution of the heme to the near-UV CD in AHb2 compared to AHb1 suggests a weaker heme-protein association in AHb2. Our data corroborate the structural diversity of AHb1 and AHb2 and confirm the leghemoglobin-like structural properties of AHb2.

Highlights

Hemoglobins (Hbs) represent a large family of globular proteins that exist in most organisms, from bacteria to higher eukaryotes [1], and participate in a broad variety of biological functions
We expand upon previous biophysical studies on these isoforms, focusing on their oligomeric states and circular dichroism (CD) characteristics
The oligomeric state of recombinant AHb1 and AHb2 in solution was analyzed by size exclusion chromatography (SEC), since it is known that the quaternary structure affects the function of many hemoglobins

Summary

Introduction

Hemoglobins (Hbs) represent a large family of globular proteins that exist in most organisms, from bacteria to higher eukaryotes [1], and participate in a broad variety of biological functions. Most of these functions are tightly associated with the prosthetic heme iron reactivity towards small ligands, especially oxygen. Symbiotic hemoglobins possess properties of oxygen transport hemoglobins as they are present in millimolar concentrations, have moderate affinity for oxygen (Kd ≈ 50 nM), a quite rapid dissociation rate constant for oxygen (~5–15 s−1), and are pentacoordinate [3] These properties facilitate their function in transporting oxygen in the root nodules of plants for symbiotic nitrogen fixation. A remarkable feature of the nsHbs is the heme hexacoordination resulting from the presence of the proximal F8 and the distal E7 histidine residues that occupy the fifth and sixth coordination position, respectively, of the heme iron

Methods

Results

Conclusion