Defense Date

1992

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

James Terner

Abstract

Chloroperoxidase is an enzyme that exhibits spectroscopic and structural properties similar to cytochrome P-450. Chloroperoxidase is studied using resonance Raman spectroscopy to characterize the reaction intermediates of the physiological mechanism, known as compounds I and II. Compound I is formed by a two electron oxidation of the resting enzyme and contains an Fe(IV) porphyrin ℼ cation radical. A one electron reduction of compound I produces the compound II intermediate which contains an oxy-ferryl [Fe(IV)=O] iron heme.

Chloroperoxidase is a heme enzyme of substantial interest because of its structural similarity to cytochrome P-450 and because of its diverse reactivity. Chloroperoxidase can function as a peroxidase, catalase, haloperoxidase and to some extent as a monooxygenase. Chloroperoxidase is excreted by the mold, Caldariomyces fumago and contains the iron protoporphyrin IX prosthetic group. From previous spectroscopic data, it has been determined that native chloroperoxidase is a penta-coordinate heme with a cysteine thiolate axial ligand.

The reaction intermediates of chloroperoxidase, compounds I and II, are among the least stable of the known peroxidase intermediates. However, they can be stabilized somewhat by avoiding the use of hydrogen peroxide as the oxidant. Because of catalase activity of this enzyme, hydrogen peroxide can act as both oxidant and substrate causing the rapid turnover of the enzyme. For the generation of the chloroperoxidase intermediates, the enzyme is mixed with an equal volume of oxidant in a Ballou four jet mixer fed by two 100 ml syringes which produces a continuous jet of newly formed intermediate. Compound I was formed by mixing the enzyme with a 15 fold excess of peracetic acid and compound II was formed by premixing the enzyme with a 100 fold excess of a substrate, ascorbic acid, then mixing with a 30 fold excess of peracetic acid.

The observed resonance Raman frequencies of the chloroperoxidase intermediates are similar to those observed for horseradish peroxidase, however there are a number of reproducible differences in frequencies due to differences in ground state symmetry or axial ligation. The in-plane skeletal modes in the resonance Raman spectrum of compound II can be assigned as follows: v10 at 1645 cm^-¹, v₃₇ at 1606 cm^-¹, v₂ at 1582 cm^-¹, v₃₈ or v¹₁₁ at 1554 cm^-¹, v₃ at 1511 cm^-¹, v₂₈ at 1476 cm^-¹, vinyl =CH₂ wags at 1345 and 1434 cm^-¹, v₂₀ or v₂₉ at 1396 cm^-¹, and v₄ at 1374 cm^-¹. These assignments are close to those previously reported for horseradish peroxidase compound II. Band assignments for compound I are v₁₀ at 1647 cm^-¹, v37 at 1619 cm^-¹, v₂ at 1589 cm^-¹ and v₄ at 1358 cm^-¹. The band at 1647 cm^-¹ is depolarized, whereas, the bands at 1619 and 1589 cm^-¹ are polarized. The oxy ferryl [Fe(IV)=O] frequency has been observed at approximately 790 cm-¹ in horseradish peroxidase. In chloroperoxidase, two bands at 790 and 753 cm^-¹ are present in both compounds I and II resonance Raman spectra. Upon ¹⁸O-substitution these bands shift approximately 30 cm^-¹ as predicted by simple force constant calculations.

Comments

Scanned, with permission from the author, from the original print version, which resides in University Archives.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

10-2-2018

Included in

Chemistry Commons

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