Publication Date

1993

Document Type

Dissertation/Thesis

First Advisor

Baker, Gary M.

Degree Name

M.S. (Master of Science)

Department

Department of Chemistry

LCSH

Cytochrome oxidase||Catalysts||Enzymes

Abstract

Two separate optical conformers that are referred to as "430 nm" and "414 nm" are known for cytochrome c oxidase. Another two separate conformers are observed upon the binding of cyanide and are referred to as "rapid" or "slow." These optical and kinetic conformers are found not to be linearly correlated, suggesting two different structural events at the catalytic core. The binding of formate to the catalytic core causes an apparent blue shift in the Soret peak, indicating 430 -> 414 nm conversion. The binding of cyanide is slowed due to the binding of formate. Formate binding is examined at concentrations varying from 0.1 to 65 mM (5 μM heme a). At the lower concentrations (≤ 5 mM), the formate binding curve is fit to a one-exponential equation. The higher concentrations of formate (≥ 10 mM) yield biphasic binding curves, even though an isosbestic point is seen in the shifting optical spectra. This biphasic response at higher concentrations of formate is observed even when the cyanide binding curve (for the solubilized enzyme, without added formate) is found to be monophasic. The distribution of the 430 and 414 nm conformers is proposed to be due to the protonation state of an amino acid residue in the proximity of the catalytic core (Papadopoulos et al., Biochemistry 30, 840-850), suggesting that the formate concentration can alter the protonation state of the amino acid residue by stimulation of proton uptake into the catalytic core. We propose that anionic formate diffuses into the catalytic core in the proton- ated form, which then loses its proton to a site that induces the 430 -> 414 nm conver- sion. A two-step kinetic mechanism showing the effects of added formate is proposed. The first and second steps both involve two optically identical forms of the enzyme undergoing 430 -> 414 nm conversion, which is required by the isosbestic point. The K[sub D]’s for the first and second steps are calculated as 3.1 and 27.9 mM, respectively. However, the apparent equilibrium dissociation constant, K[sub D]^(app), for the entire mechanism is calculated as 0.34 mM. This small value for the K[sub D]^(app) in comparison to the K[sub D] for the separate steps is found to be not consistent with the proposed mechanism. A second mechanism is then proposed. This mechanism is found to be much more acceptable according to the data at hand. The two-step mechanism states that the 430 -> 414 nm conversion occurs only in the first step. The second step is an isomerization step, involving only the 414 nm form, where the two forms of the enzyme are optically identical, again due to the isosbestic point. The two steps of the mechanism account for the 2 phases of formate binding and transition to monophasic binding at lower concentrations of formate.

Comments

Includes bibliographical references (pages [68]-70)

Extent

v, 72 pages

Language

eng

Publisher

Northern Illinois University

Rights Statement

In Copyright

Rights Statement 2

NIU theses are protected by copyright. They may be viewed from Huskie Commons for any purpose, but reproduction or distribution in any format is prohibited without the written permission of the authors.

Media Type

Text

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