Manon Couture

A novel two-over-two alpha-helical sandwich fold is characteristic of the truncated hemoglobin family.

Small hemoproteins displaying amino acid sequences 20–40 residues shorter than (non‐)vertebrate hemoglobins (Hbs) have recently been identified in several pathogenic and non‐pathogenic unicellular organisms, and named ‘truncated hemoglobins’ (trHbs). They have been proposed to be involved not only in oxygen transport but also in other biological functions, such as protection against reactive nitrogen species, photosynthesis or to act as terminal oxidases. Crystal structures of trHbs from the ciliated protozoan Paramecium caudatum and the green unicellular alga Chlamydomonas eugametos show that the tertiary structure of both proteins is based on a ‘two‐over‐two’ α‐helical sandwich, reflecting an unprecedented editing of the classical ‘three‐over‐three’ α‐helical globin fold. Based on specific Gly–Gly motifs the tertiary structure accommodates the deletion of the N‐terminal A‐helix and replacement of the crucial heme‐binding F‐helix with an extended polypeptide loop. Additionally, concerted structural modifications allow burying of the heme group and define the distal site, which hosts a TyrB10, GlnE7 residue pair. A set of structural and amino acid sequence consensus rules for stabilizing the fold and the bound heme in the trHbs homology subfamily is deduced.

A Cooperative Oxygen Binding Hemoglobin from Mycobacterium tuberculosis STABILIZATION OF HEME LIGANDS BY A DISTAL TYROSINE RESIDUE

The homodimeric hemoglobin (HbN) fromMycobacterium tuberculosis displays an extremely high oxygen binding affinity and cooperativity. Sequence alignment with other hemoglobins suggests that the proximal F8 ligand is histidine, the distal E7 residue is leucine, and the B10 position is occupied by tyrosine. To determine how these heme pocket residues regulate the ligand binding affinities and physiological functions of HbN, we have measured the resonance Raman spectra of the O2, CO, and OH− derivatives of the wild type protein and the B10 Tyru2009→u2009Leu and Phe mutants. Taken together these data demonstrate a unique distal environment in which the heme bound ligands strongly interact with the B10 tyrosine residue. The implications of these data on the physiological functions of HbN and another heme-containing protein, cytochrome c oxidase, are considered.

Chlamydomonas Chloroplast Ferrous Hemoglobin HEME POCKET STRUCTURE AND REACTIONS WITH LIGANDS

We report the optical and resonance Raman spectral characterization of ferrous recombinantChlamydomonas LI637 hemoglobin. We show that it is present in three pH-dependent equilibrium forms including a 4-coordinate species at acid pH, a 5-coordinate high spin species at neutral pH, and a 6-coordinate low spin species at alkaline pH. The proximal ligand to the heme is the imidazole group of a histidine. Kinetics of the reactions with ligands were determined by stopped-flow spectroscopy. At alkaline pH, combination with oxygen, nitric oxide, and carbon monoxide displays a kinetic behavior that is interpreted as being rate-limited by conversion of the 6-coordinate form to a reactive 5-coordinate form. At neutral pH, combination rates of the 5-coordinate form with oxygen and carbon monoxide were much faster (>107 μm −1 s−1). The dissociation rate constant measured for oxygen is among the slowest known, 0.014 s−1, and is independent of pH. Replacement of the tyrosine 63 (B10) by leucine or of the putative distal glutamine by glycine increases the dissociation rate constant 70- and 30-fold and increases the rate of autoxidation 20- and 90-fold, respectively. These results are consistent with at least two hydrogen bonds stabilizing the bound oxygen molecule, one from tyrosine B10 and the other from the distal glutamine. In addition, the high frequency (232 cm−1) of the iron-histidine bond suggests a structure that lacks any proximal strain thus contributing to high ligand affinity.