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 O 2 combination rate, but almost no change in the O 2 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 O 2 and CO, as evidenced by the increase of the Fe-O 2 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 O 2 and CO. Also, the rate of O 2 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 O 2 binding and stabilization.
|Titolo:||The roles of Tyr(CD1) and Trp(G8) in Mycobacterium tuberculosis truncated hemoglobin O in ligand binding and on the heme distal site architecture|
|Autori interni:||BOLOGNESI, MARTINO|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Data di pubblicazione:||2007|
|Digital Object Identifier (DOI):||10.1021/bi7010288|
|Appare nelle tipologie:||01 - Articolo su periodico|