Truncated hemoglobin O (trHbO) is one of two trHbs in
Mycobacterium tuberculosis.Remarkably, trHbO possesses two novel distal residues, in addition to the B10 tyrosine, that may beimportant in ligand binding. These are the CD1 tyrosine and G8 tryptophan. Here we investigate thereactions of trHbO and mutants using stopped-flow spectrometry, flash photolysis, and UV-enhancedresonance Raman spectroscopy. A biphasic kinetic behavior is observed for combination and dissociationof O
2 and CO that is controlled by the B10 and CD1 residues. The rate constants for combination (<1.0
M
-1 s
-1) and dissociation (<0.006 s
-1) of O
2 are among the slowest known, precluding transport ordiffusion of O
2 as a major function. Mutation of CD1 tyrosine to phenylalanine shows that this groupcontrols ligand binding, as evidenced by 25- and 77-fold increases in the combination rate constants forO
2 and CO, respectively. In support of a functional role for G8 tryptophan, UV resonance Raman indicatesthat the
(2,1) dihedral angle for the indole ring increases progressively from approximately 93
to at least100
in going sequentially from the deoxy to CO to O
2 derivative, demonstrating a significantconformational change in the G8 tryptophan with ligation. Remarkably, protein modeling predicts a networkof hydrogen bonds between B10 tyrosine, CD1 tyrosine, and G8 tryptophan, with the latter residues beingwithin hydrogen bonding distance of the heme-bound ligand. Such a rigid hydrogen bonding networkmay thus represent a considerable barrier to ligand entrance and escape. In accord with this model, wefound that changing CD1 or B10 tyrosine for phenylalanine causes only small changes in the rate of O
2dissociation, suggesting that more than one hydrogen bond must be broken at a time to promote ligandescape. Furthermore, trHbO-CO cannot be photodissociated under conditions where the CO derivativeof myoglobin is extensively photodissociated, indicating that CO is constrained near the heme by thehydrogen bonding network.
