Journal:JBIC:8

A hydrogen-bonding network formed by the B10-E7-E11 residues of a truncated hemoglobin from Tetrahymena pyriformis is critical for stability of bound oxygen and nitric oxide detoxification

Jotaro Igarashi, Kazuo Kobayashi and Ariki Matsuoka^[1]

Molecular Tour
A wide diversity of both structure and function has been discovered in the study of hemoglobins (Hbs) from many species. Hbs transport oxygen in the red blood cells of higher organisms. Even though oxygen molecules can diffuse into cells, unicellular organisms also have Hb-like molecules. Here, we consider the term 'hemoglobins' to include such molecules. Three classes of Hb have been found in unicellular organisms. First, single-domain globins are comprised of three-over-three (3/3) α-helical folds, as is myoglobin (Mb). Second, flavohemoglobins are distinguished by the presence of an N-terminal globin domain and an additional C-terminal FAD-containing reductase region. Finally, truncated Hbs (trHbs) have been discovered recently and are widely distributed in unicellular organisms.

Truncated hemoglobins, also known as 2/2 hemoglobins, can be further classified into three different groups (I, II, and III). Genomic sequences of bacteria, cyanobacteria, and plants indicate that trHbs are rather common. Group I, Group II, and Group III trHbs have distinct phylogenetic trees and show different ligand-binding properties. The Group I trHb of the ciliated protozoan Tetrahymena pyriformis (Tp trHb) was first discovered by Keilin and Ryley in 1953.

It is known that trHbs exist in ciliates of the Tetrahymena group, but trHb structure and function remain poorly understood. To investigate trHb function with respect to stability of bound oxygen and protein structure, we measured the oxygen binding kinetics of Tetrahymena pyriformis trHb, and determined the crystal structure of the protein.

The three-dimensional structure of an Fe(II)-O2 complex of Tp trHb was determined at 1.73 Å resolution (3aq9). Tyr25 (B10) and Gln46 (E7) were hydrogen-bonded to a heme-bound dioxygen molecule. Tyr25 donated a hydrogen bond to the terminal oxygen atom, whereas Gln46 hydrogen-bonded to the proximal oxygen atom. Furthermore, Tyr25 was hydrogen-bonded to the Gln46 and Gln50 (E11) residues.

The O₂ association and dissociation rate constants of T. pyriformis trHb were 5.5 μM^-1 s^-1, and 0.18 s^-1, respectively. The oxygen affinity was determined to be 33 nM. The autooxidation rate constant was 3.8 x 10^-3 h^-1. These values are similar to those of HbN from Mycobacterium tuberculosis.

Mutations:

• Mutation at Tyr25: Wildtype Y25 and mutant Y25F together and animation of this scene.
• Mutation at Gln46: Wildtype Q46 and mutant Q46E together (animation)
• Mutation at Gln50 increased the O₂ dissociation and autooxidation rate constants, and partly disrupted the hydrogen-bonding network.

An Fe(III)-H2O complex of Tp trHb was formed following reaction of the Fe(II)-O2 complex of Tp trHb, in a crystal state, with nitric oxide. This suggests that Tp trHb functions in nitric oxide detoxification.

PDB reference: Crystal structure of truncated hemoglobin from Tetrahymena pyriformis, Fe(II)-O2 form 3aq5; Crystal structure of truncated hemoglobin from Tetrahymena pyriformis, Fe(III) form 3aq6; Crystal structure of truncated hemoglobin from Tetrahymena pyriformis, Y25F mutant, Fe(III) form 3aq7; Crystal structure of truncated hemoglobin from Tetrahymena pyriformis, Q46E mutant, Fe(III) form 3aq8; Crystal structure of truncated hemoglobin from Tetrahymena pyriformis, Q50E mutant, Fe(III) form 3aq9.