A Cooperative Oxygen Binding Hemoglobin from Mycobacterium tuberculosis

Abstract

The homodimeric hemoglobin (HbN) from Mycobacterium 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 O 2 , CO, and OH ؊ derivatives of the wild type protein and the B10 Tyr 3 Leu 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.

Deciphering the Structural Role of Histidine 83 for Heme Binding in Hemophore HasA

“…S2C). All of these findings are consistent with an hydroxidebound form (36,(61)(62)(63). Two broad and exchangeable signals, missing at acidic pH, are observable at alkaline pH (a major one at 64.7 ppm and a minor one at 70.5 ppm at pH 9.9 and 303 K) (Fig.…”

NO Binding Induced Conformational Changes in a Truncated Hemoglobin from Mycobacterium tuberculosis

“…On the other hand, it shifts to a higher frequency when the phenolic oxygen of Tyr forms an H-bond with a proton donor. The ferric heme of the aquo-met derivative of HbN is coordinated by a water molecule in the distal position at neutral pH (18). The Y 8a mode at 1616 cm -1 identified in the spectrum of the aquomet derivative was assigned to B10 Tyr33 as discussed earlier (Figure 5a).…”

“…The phenolic oxygen atom of the B10 Tyr side chain forms an H-bond with the nitrogen atom of the side chain of Gln58 at the E11 position, which anchors the B helix to the E helix and creates a polar environment for the heme ligands, as shown in Figures 1 and 6. Resonance Raman spectroscopic results suggest that B10 Tyr33 forms H-bonds with various heme ligands, including CO and O 2 in the ferrous derivatives, and hydroxide in the ferric derivatives (18). The H-bonding interaction in the oxy derivative is confirmed by the crystallographic data, in which the phenolic hydroxide of B10 Tyr33 forms H-bonds with both oxygen atoms of the heme-bound dioxygen (Figure 1).…”

“…It was proposed that this unique heme environment stabilizes the NO and prevents autoreduction of the heme, which is very important for nitrophorin to perform its physiological function (33). Intriguingly, the Fe-NO stretching and Fe-N-O bending frequencies of nitrophorin determined by resonance Raman measurements were identical to those of the NO derivative of HbN (38), despite the fact that the distal pocket of nitrophorin is hydrophobic in contrast to the hydrophilic pocket of HbN (18,19). In contrast, horse heart myoglobin, which has a distal His at the E7 position that stabilizes the heme-bound NO, has a much more intense Fe-N-O bending mode with a lower frequency (572 cm -1 ) and a higher Fe-NO stretching frequency (596 cm -1 ) (39).…”

“…Like mammalian globins, HbN binds various heme ligands reversibly (18,19). Resonance Raman studies suggest that these heme-bound ligands are stabilized by a distal Tyr residue at the B10 position (18), which has been subsequently confirmed by the recent crystallographic data (20). Biochemical and biophysical studies suggest that the oxygen affinity of HbN is extremely high and its biological function may be involved in NO detoxification (18,19,21).…”