A Leucine Residue “Gates” Solvent but Not O2Access to the Binding Pocket of Phascolopsis gouldiiHemerythrin

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). UV-visible and resonance Raman spectra showed that the di-iron sites in these L98 X Hrs are very similar to those in the wild type protein, and several of the L98 X hemerythrins formed stable oxy adducts. Despite the apparently tight packing in the pocket, all of the L98 X 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 L98 X 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.