A Leucine Residue "Gates" Solvent but Not O2
Access to the Binding Pocket of Phascolopsis gouldii
Hemerythrin*
Christopher S.
Farmer
,
Donald M.
Kurtz Jr.§,
Robert S.
Phillips,
Jingyuan
Ai¶, and
Joann
Sanders-Loehr¶
From the Department of Chemistry and Center for
Metalloenzyme Studies, University of Georgia, Athens, Georgia
30602-2556 and the ¶ Oregon Graduate Institute for Science and
Technology, Portland, Oregon 97291-1000
A leucine residue, Leu-98, lines the
O2-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 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
O2 association rate constants within a factor of 3 of the wild type value. Similarly, the O2 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 O2 dissociation rate constant and a large
D2O effect on this rate, which are attributed to a
hydrogen-bonding interaction between the Tyr-98 hydroxyl and the bound
O2. 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 O2, but is evident for solvent.
*
This work was supported by grants GM40388 (D.M.K.), and
GM18865 (J.S.-L.) from the National Institutes of Health.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF22052.
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