The O(2) binding pocket of myohemerythrin: role of a conserved leucine.
A conserved O(2) binding pocket residue in Phascolopsis gouldii myohemerythrin (myoHr), namely, L104, was mutated to several other residues, and the effects on O(2) association and dissociation rates, O(2) affinity, and autoxidation were examined. The L104V, -F, and -Y myoHrs formed stable O(2) adducts whose UV-vis and resonance Raman spectra closely matched those of wild-type oxymyoHr. The L104V mutation produced only minimal effects on either O(2) association or dissociation, whereas the L104F and -Y mutations resulted in 100-300-fold decreases in both O(2) association and dissociation rates. These decreases are attributed to introduction of steric restrictions into the O(2) binding pocket, which are not present in either wild-type or L104V myoHrs. The failure to observe increased O(2) association or dissociation rates for L104V indicates that the side chain of leucine at position 104 does not sterically "gate" O(2) entry into or exit from the binding pocket in the rate-determining step(s). L104V myoHr autoxidized approximately 3 times faster than did wild type, whereas L104T autoxidized >10(6) times faster than did wild type. The latter large increase is attributed to increased side chain polarity, thereby increasing water occupancy in the oxymyoHr binding pocket. These results indicate that L104 contributes a hydrophobic barrier that restricts water entry into the oxymyoHr binding pocket. Thus, a leucine at position 104 in myoHr appears to have the optimal combination of size and hydrophobicity to facilitate O(2) binding while simultaneously inhibiting autoxidation.
Department of Chemistry, University of Georgia, Athens 30602-2556, USA.
J
J
Xiong
R S
RS
Phillips
D M
DM
Kurtz
S
S
Jin
J
J
Ai
J
J
Sanders-Loehr
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017124:Conserved Sequence; D017931:DNA Primers / Q000235:genetics; D006422:Hemerythrin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D066298:In Vitro Techniques; D007700:Kinetics; D007930:Leucine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009348:Nematoda / Q000235:genetics / Q000378:metabolism; D010100:Oxygen / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman
D017931:DNA Primers; D006422:Hemerythrin; D011994:Recombinant Proteins; C008571:myohemerythrin; D007930:Leucine; D010100:Oxygen