A leucine residue, Leu-98, lines the O 2 -binding pocket in all known hemerythrins. Leu-98 in recombinant Phascolopsis gouldii hemerythrin, was mutated to several other residues of varying sizes (Ala, Val), polarities (Thr, Asp, Asn), and aromaticities (Phe, Tyr, Trp). UVvisible 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 formed stable oxy adducts. Despite the apparently tight packing in the pocket, all of the L98X Hrs except for L98W, had second order O 2 association rate constants within a factor of 3 of the wild type value. Similarly, the O 2 dissociation rate constant was essentially unaffected by substitutions of larger (Phe) or smaller (Val, Thr) residues for Leu-98. L98Y Hr showed a 170-fold decrease in the O 2 dissociation rate constant and a large D 2 O effect on this rate, which are attributed to a hydrogen-bonding interaction between the Tyr-98 hydroxyl and the bound O 2 . Significant increases in autoxidation rates were observed for all of the L98X Hrs other than X ‫؍‬ Tyr. These increases 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 hydrophobicity for inhibition of solvent access. Thus, "gating" of small molecule access to the binding pocket of Hr by Leu-98 is not evident for O 2 , but is evident for solvent.

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.

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