Li, Zhuyin (1992) Proton pumping in cytochrome c oxidase: the possible role of Cu_A in redox linkage. Dissertation (Ph.D.), California Institute of Technology. http://resolver.caltech.edu/CaltechTHESIS:09262011-094449362
Cytochrome c oxidase is the terminal enzyme of cellular respiration. It catalyzes the reduction of dioxygen to water by ferrocytochrome c. In addition, cytochrome c oxidase is an electrogenic proton pump capable of transporting up to four protons against the proton electrochemical gradient.
An extensive and accurate analysis of the Cu, Fe, Zn and Mg contents in bovine heart cytochrome c oxidase was carried out by direct current plasma atomic emission spectrophotometer. The results confirmed a stoichiometry of 5Cu/4Fe/2Zn/2Mg per dimer. Seven enzyme preparations treated by different methods were analyzed to examine the nature of Cu_X. The results obtained suggested that Cux may reside in subunit III and plays a structural role in enzyme dimerization.
pHMB-modification of cytochrome c oxidase and heat-treatment of the oxidase have previously been shown to have a profound effect on the dioxygen reduction and proton pumping activities of the enzyme. The intrinsic reduction potentials and the midpoint reduction potential of the "Cu_A" site in both of these modified oxidases have been measured under various conditions in order to clarify the intramolecular electron transfer pathways in these systems. The study reveals that the Cu_A intrinsic reduction potential decreases by almost 150 mV upon pHMB modification, whereas it increases by 100 mV upon heat treatment. In addition, the redox interactions between Cu_A and the remaining metal centers are perturbed upon Cu_A modification.
DCCD-modification and subunit III-depletion of cytochrome c oxidase decreased the observed H+/e- stoichiometry in proton pumping to 0.5 during enzyme turnover under coupled conditions. The effects of these treatments on the optical and EPR spectra, and the reduction potentials of Cu_A. have been examined. DCCD-modification distorts the Cu_A ligand structure, while subunit III-depletion destabilizes the Cu_A structure leading ultimately to the displacement of one of the cysteine ligands. The intrinsic reduction potential of Cu_A is not significantly altered in the two modified oxidases. However, the redox interaction between Cu_A and cytochrome a becomes dramatically different: in the absence of subunit III, this redox interaction is essentially eliminated, whereas in the DCCD-modified enzyme, it becomes highly anticooperative. The results of this study implicate Cu_A as a component of the proton pumping machinery of the enzyme, and suggest that subunit III plays an important allosteric role in communicating the redox changes at the Cu_A site to the proton translocating element(s) of the proton pumping machinery during turnover.
The strong magnetic interaction between Fe_(a3) and Cu_B in the resting form of cytochrome c oxidase is thought to be mediated by a bridging ligand. The identity of this bridging ligand has been proposed to be an imidazole from a histidine residue, cysteine thiolate sulphur, an oxygen from hydroxide or μ-oxo anions. However, recent EXAFS experiments on a sample of oxidase isolated and purified in Cl^--free buffer solutions revealed no (S, Cl) scattering in Fe EXAFS of the resting oxidase, thus suggesting Cl^-. To confirm this finding, we have dialyzed samples of reduced oxidase against Cl^--free buffer in the presence of chelex resin, and have examined the spectroscopic and ligand-binding properties of the reoxidized enzyme. The dialyzed/oxygenated oxidase exhibits rapid and homogeneous binding kinetics. Upon re-addition of Cl^- to the dialyzed/oxygenated oxidase, the CN^-binding slows down and becomes heterogeneous. The anions F^-, Cl^-and Br^- all exhibit notable effects on the Soret absorption of the dialyzed/oxygenated oxidase. Similarly the EPR spectra of CI^- or Br^-substituted oxidase are similar to native oxidase. Both F^--substituted oxidase and dialyzed / oxygenated oxidase exhibit complex EPR spectra. On the basis of these experiments, we conclude that the bridging ligand must be Cl^- in resting cytochrome c oxidase.
|Item Type:||Thesis (Dissertation (Ph.D.))|
|Degree Grantor:||California Institute of Technology|
|Division:||Chemistry and Chemical Engineering|
|Thesis Availability:||Restricted to Caltech community only|
|Defense Date:||1 October 1991|
|Default Usage Policy:||No commercial reproduction, distribution, display or performance rights in this work are provided.|
|Deposited By:||John Wade|
|Deposited On:||26 Sep 2011 18:02|
|Last Modified:||26 Dec 2012 04:38|
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