2QRW

Crystal structure of Mycobacterium tuberculosis trHbO WG8F mutant

Summary for 2QRW

• Entry DOI: 10.2210/pdb2qrw/pdb
• Related: 1ngk
• Descriptor: Hemoglobin-like protein HbO, CYANIDE ION, SULFATE ION, ... (5 entities in total)
• Functional Keywords: truncated hemoglobin fold, alpha helix, heme, hydroxylation, iron, membrane, metal-binding, oxygen transport, transport, transport protein
• Biological source: Mycobacterium tuberculosis
• Total number of polymer chains: 12
• Total formula weight: 189113.40

Authors

Milani, M.,Bolognesi, M. (deposition date: 2007-07-30, release date: 2007-11-06, Last modification date: 2023-08-30)

Primary citation

Ouellet, H.,Milani, M.,LaBarre, M.,Bolognesi, M.,Couture, M.,Guertin, M.
The Roles of Tyr(CD1) and Trp(G8) in Mycobacterium tuberculosis Truncated Hemoglobin O in Ligand Binding and on the Heme Distal Site Architecture
Biochemistry, 46:11440-11450, 2007

PubMed Abstract: The crystal structure of the cyano-met form of Mt-trHbO revealed two unusual distal residues Y(CD1) and W(G8) forming a hydrogen-bond network with the heme-bound ligand [Milani, M., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5766-5771]. W(G8) is an invariant residue in group II and group III trHbs and has no counterpart in other globins. A previous study reported that changing Y(CD1) for a Phe causes a significant increase in the O2 combination rate, but almost no change in the O2 dissociation rate [Ouellet, H., et al. (2003) Biochemistry 42, 5764-5774]. Here we investigated the role of the W(G8) in ligand binding by using resonance Raman spectroscopy, stopped-flow spectrophotometry, and X-ray crystallography. For this purpose, W(G8) was changed, by site-directed mutagenesis, to a Phe in both the wild-type protein and the mutant Y(CD1)F to create the single mutant W(G8)F and the double mutant Y(CD1)F/W(G8)F, respectively. Resonance Raman results suggest that W(G8) interacts with the heme-bound O2 and CO, as evidenced by the increase of the Fe-O2 stretching mode from 559 to 564 cm-1 and by the lower frequency of the Fe-CO stretching modes (514 and 497 cm-1) compared to that of the wild-type protein. Mutation of W(G8) to Phe indicates that this residue controls ligand binding, as evidenced by a dramatic increase of the combination rates of both O2 and CO. Also, the rate of O2 dissociation showed a 90-1000-fold increase in the W(G8)F and Y(CD1)F/W(G8)F mutants, that is in sharp contrast with the values obtained for the other distal mutants Y(B10)F and Y(CD1)F [Ouellet, H., et al. (2003) Biochemistry 42, 5764-5774]. Taken together, these data indicate a pivotal role for the W(G8) residue in O2 binding and stabilization.

PubMed: 17887774
DOI: 10.1021/bi7010288

Experimental method

X-RAY DIFFRACTION (1.93 Å)