The O-2 binding pocket of myohemerythrin: Role of a conserved leucine

A conserved O-2 binding pocket residue in Phascolopsis gouldii myohemerythr in (myoHr), namely, L104, was mutated to several other residues, and the ef fects on Oz association and dissociation rates, O-2 affinity, and autoxidat ion were examined. The L104V, -F, and -Y myoHrs formed stable Oz adducts wh ose UV-vis and resonance Raman spectra closely matched those of wild-type o xymyoHr. The L104V mutation produced only minimal effects on either O-2 ass ociation or dissociation, whereas the L104F and -Y mutations resulted in 10 0-300-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 Oz association or dissociation rates for L104V indicates that the side chain of leucine at position 104 does not st erically "gate" O-2 entry into or exit from the binding pocket in the rate- determining step(s). L104V myoHr autoxidized approximately 3 times faster t han did wild type, whereas L104T autoxidized >10(6) times faster than did w ild type. The latter large increase is attributed to increased side chain p olarity, thereby increasing water occupancy in the oxymyoHr binding pocket. These results indicate that L104 contributes a hydrophobic barrier that re stricts water entry into the oxymyoHr binding pocket. Thus, a leucine at po sition 104 in myoHr appears to have the optimal combination of size and hyd rophobicity to facilitate O-2 binding while simultaneously inhibiting autox idation.