title: The crystal structures of Phascolopsis gouldii wild type and L98Y methemerythrins: structural and functional alterations of the O2 binding pocket.
abstract: Reported are the X-ray crystal structures of recombinant Phascolopsis gouldii methemerythrin (1.8-A resolution) and the structure of an O2-binding-pocket mutant, L98Y methemerythrin (2.1-A resolution). The L98Y hemerythrin (Hr) has a greatly enhanced O2 affinity, a slower O2 dissociation rate, a larger solvent deuterium isotope effect on this rate, and a greater resistance to autoxidation relative to the wild-type protein. The crystal structures show that the hydrophobic binding pocket of Hr can accommodate substitution of a leucyl by a tyrosyl side chain with relatively minor structural rearrangements. UV/vis and resonance Raman spectra show that in solution L98Y methemerythrin contains a mixture of two diiron site structures differing by the absence or presence of an Fe(III)-coordinated phenolate. However, in the crystal, only one L98Y diiron site structure is seen, in which the Y98 hydroxyl is not a ligand, but instead forms a hydrogen bond to a terminal hydroxo/aqua ligand to the nearest iron. Based on this crystal structure, we propose that in the oxy form of L98Y hemerythrin the non-polar nature of the binding pocket favors localization of the Y98 hydroxyl near the O2 binding site, where it can donate a hydrogen bond to the hydroperoxo ligand. The stabilizing Y98OH-O2H-interaction would account for all of the altered O2 binding properties of L98Y Hr listed above.
authors.affiliation: Department of Chemistry and Center for Metalloenzyme Studies, University of Georgia, Athens, GA 30602-2556, USA.
authors.forename: C S
authors.initials: CS
authors.lastname: Farmer
authors.forename: D M
authors.initials: DM
authors.lastname: Kurtz
authors.forename: Z J
authors.initials: ZJ
authors.lastname: Liu
authors.forename: B C
authors.initials: BC
authors.lastname: Wang
authors.forename: J
authors.initials: J
authors.lastname: Rose
authors.forename: J
authors.initials: J
authors.lastname: Ai
authors.forename: J
authors.initials: J
authors.lastname: Sanders-Loehr
mesh_terms: D000818:Animals; D001665:Binding Sites; D018360:Crystallography, X-Ray; D006422:Hemerythrin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D009348:Nematoda / Q000201:enzymology*; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman
chemical_list: D006422:Hemerythrin; D011994:Recombinant Proteins; C019004:methemerythrin; D010100:Oxygen