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The buried charge of Asp-235 in cytochrome c peroxidase (CCP) forms an important
hydrogen bond to the histidine ligand of the heme iron. The Asp-His-metal
interaction, which is similar to the catalytic triad of serine proteases, is
found at the active site of many metalloenzymes and is believed to modulate the
character of histidine as a metal ligand. We have examined the influence of this
interaction in CCP on the function, redox properties, and iron zero-field
splitting in the native ferric state and its effect on the Trp-191 free radical
site in the oxidized ES complex. Unlike D235A and D235N, the mutation D235E
introduces very little perturbation in the X-ray crystal structure of the enzyme
active site, with only minor changes in the geometry of the
carboxylate-histidine interaction and no observable change at the Trp-191 free
radical site. More significant effects are observed in the position of the helix
containing residue Glu-235. However, the small change in hydrogen bond geometry
is all that is necessary to (1) increase the reduction potential by 70 mV, (2)
alter the anisotropy of the Trp-191 free radical EPR, (3) affect the activity
and spin-state equilibrium, and (4) reduce the strength of the iron ligand field
as measured by the zero-field splitting. The changes in the redox potential with
substitution are correlated with the observed zero-field splitting, suggesting
that redox control is exerted through the heme ligand by a combination of
electrostatic and ligand field effects. The replacement of Asp-235 with Glu
appears to result in a significantly weaker hydrogen bond in which the proton
resides essentially with His-175. This hydrogen bond is nevertheless strong
enough to prevent the reorientation of Trp-191 and the conversion to one of two
low-spin states observed for D235A and D235N. The Asp-His-Fe interaction is
therefore as important in defining the redox properties and imidazolate
character of His-175 as has been proposed, yet its most important role in
peroxidase function may be to correctly orient Trp-191 for efficient coupling of
the free radical to the heme and to maintain a high-spin 5-coordinate heme
center.
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