The roles of Tyr(CD1) and Trp(G8) in Mycobacterium tuberculosis truncated hemoglobin O in ligand binding and on the heme distal site architecture.
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.
Ouellet
Hugues
H
Department of Biochemistry and Microbiology, Laval University, Quebec, Canada, G1K 7P4.
Milani
Mario
M
LaBarre
Marie
M
Bolognesi
Martino
M
Couture
Manon
M
Guertin
Michel
M
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites; D018360:Crystallography, X-Ray; D017931:DNA Primers; D006418:Heme / Q000737:chemistry*; D006454:Hemoglobins / Q000737:chemistry; D006860:Hydrogen Bonding; D008024:Ligands; D016296:Mutagenesis; D013059:Spectrum Analysis, Raman; D054793:Truncated Hemoglobins / Q000737:chemistry* / Q000235:genetics; D014364:Tryptophan; D014443:Tyrosine
D001426:Bacterial Proteins; D017931:DNA Primers; D006454:Hemoglobins; D008024:Ligands; D054793:Truncated Hemoglobins; C451738:hemoglobin O, Mycobacterium tuberculosis; D014443:Tyrosine; D006418:Heme; D014364:Tryptophan