Title

Active Site Coordination Chemistry of the Cytochrome c Peroxidase Asp235Ala Variant: Spectroscopic and Functional Characterization

Document Type

Article

Publication Date

6-1-1994

Abstract

Asp235 in yeast cytochrome c peroxidase forms a hydrogen bond with His175, the proximal histidyl residue, that has been suggested to be important in determining the electronic properties of the heme iron and that may be involved in stabilizing the higher oxidation states of the peroxidase that form transiently during catalysis. The current study employs 1H and 15N-NMR spectroscopy to study the electronic properties of and the effects of pH on the active site of the Asp235Ala variant. This variant exhibits three spectroscopic species between pH 5 and 9: a high-spin species that forms at low pH and two low-spin species that form successively at higher pH. Nevertheless, the activity of the variant exhibits a pH dependence virtually identical to that of the wild-type protein, though the activity of the variant is 3 orders of magnitude lower at all values of pH between pH 5 and 8.5. These findings suggest that the spin state and coordination environment of the heme iron in cytochrome c peroxidase do not dictate the rate of substrate (cytochrome c) oxidation. Binding of cyanide to the variant enzyme results in formation of a single species as detected by NMR spectroscopy. Analysis of high-resolution 1D and 2D 1H-NMR and 15N-NMR spectra of the cyanide adduct has permitted characterization of the properties of this derivative and the strength of the proximal ligand bond to the heme iron. Disruption of the hydrogen bond between the proximal histidine and Asp235 that exists in the wild-type enzyme dramatically reduces the strength of the interaction between the proximal ligand and the iron; this effect combined with concurrent changes in the distal heme-binding pocket accounts for the increase in reduction potential reported for the Fe3+/Fe2+ couple. The catalytic consequences of the structural and electronic properties of the variant elucidated in this study are discussed. © 1994, American Chemical Society. All rights reserved.

Publication Source (Journal or Book title)

Biochemistry

First Page

7819

Last Page

7829

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