Cytochrome oxidase, the terminal oxidase of the electron transport chain, is classically known to be a membrane bound protein of a molecular weight close to 200,000 Daltons which contains two copper atoms (Cu(,D) and Cu(,U)) and two heme groups, cytochromes a and a(,3). The enzyme is found in aerobic organisms where its function is to catalyze the reduction of oxygen to water and to promote oxidative phosphorylation to satisfy the energy requirements of the cell.
The purpose of this thesis is to define the oxidative-reductive (redox) intermediates of the enzyme. Physico-chemical methods are used to obtain information on the structural and valence characteristics, the electronic states, and the redox properties of the four metals involved in catalysis. Methods such as magnetic susceptibility, electronic absorption spectroscopy, electron paramagnetic resonance (EPR), and magnetic circular dichroism (MCD) have been used to monitor several types of redox intermediates. Redox titrations have been performed under different pH conditions, with various ligands, with several mediators, under variable salt concentrations, and in the presence of mitochondrial metabolites such as ATP. The low temperature (77(DEGREES)K) MCD spectra of "mixed-valence" cytochrome oxidase have been recorded.
By using multiple linear regression of the spectral data and computer simulations of the redox titrations monitored by optical EPR, and MCD spectroscopies the absolute and relative redox potentials of the enzyme under various conditions have been obtained. With these methods the redox intermediates of cytochrome oxidase can be defined.
The results indicate that for the reductive titration in the presence of cytochrome c at pH 7.4 (15 C) the potentials are:
cytochrome a = cytochrome a(,3) = Cu(,U) = 314 mV,
Cu(,D) = 272 mV.
At pH 8.5 (15 C), the potential of cytochrome a(,3) is lowered by 13 mV and the potential Cu(,U) is lowered by 38 mV with respect to cytochrome a. In the presence of inhibitors which stabilize the oxidized form of cytochrome a(,3) (azide and formate), the redox potentials of cytochrome a(,3) and Cu(,U) are lowered by 77 mV with respect to cytochrome a. In the presence of inhibitors which stabilize the reduced form of cytochrome a(,3) (carbon monoxide), the redox potential of cytochrome a(,3) is increased by 100 mV. No evidence of "heme-heme interaction" is observed at equilibrium.
The relative potentials measured at 15 C and at cryogenic temperatures indicate that electron rearrangements are occurring among the redox centers of the partially reduced enzyme samples during freezing of the EPR samples. A model for the bridging between cytochrome a(,3) and the Cu(,U) center is presented which explains the three types of high-spin signals that are observed.
This thesis clarifies the assignments of the electronic and magnetic characteristics which each redox center exhibits, defines the absolute and relative potentials associated with each metal center, and discriminates between the existing models of the mechanism of cytochrome oxidase in order to establish the oxidative-reductive mechanism of the enzyme.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/15549 |
| Date | January 1980 |
| Creators | GARCIA-INIGUEZ, LUCIA RAFAELA |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | application/pdf |
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