A leucine residue "gates" solvent but not O-2 access to the binding pocketof Phascolopsis gouldii hemerythrin

A leucine residue, Leu-98, Lines the O-2-binding pocket in all known hemery thrins. Leu-98 in recombinant Phascolopsis gouldii hemerythrin, was mutated to several other residues of varying sizes (Ala, Val), polarities (Thr, As p, Asn), and aromaticities (Phe, Tyr, Trp). UV-visible and resonance Raman spectra showed that the di-iron sites in these L98X Hrs are very similar to those in the wild type protein, and several of the L98X hemerythrins forme d stable oxy adducts, Despite the apparently tight packing in the pocket, a ll of the L98X Hrs except for L98W, had second order O-2 association rate c onstants within a factor of 3 of the wild type value. Similarly, the O-2 di ssociation rate constant was essentially unaffected by substitutions of lar ger (Phe) or smaller (Val, Thr) residues for Leu-98, L98Y Hr showed a 170-f old decrease in the O-2 dissociation rate constant and a large D2O effect o n this rate, which are attributed to a hydrogen-bonding interaction between the Tyr-98 hydroxyl and the bound O-2. Significant increases in autoxidati on rates were observed for all of the L98X Hrs other than X = Tyr. These in creases in autoxidation rates are attributed to increased solvent access to the binding pocket caused by inefficient packing (Phe), smaller size (Val, Ala), or increased polarity (Thr, Asp, Asn) of the residue 98 side chain. A leucine at position 98 appears to have the optimal size, shape, and hydro phobicity for inhibition of solvent access. Thus, "gating" of small molecul e access to the binding pocket of Hr by Leu-98 is not evident for O-2, but is evident for solvent.