The Nature and Distribution of the Metal Centers in Cytochrome C Oxidase

Author: Brudvig, Gary Wayne

Year: 1981

Degree: Dissertation (Ph.D.)

Advisor: Chan, Sunney I.

Committee Member: Unknown, Unknown

Option: Chemistry

DOI: 10.7907/yms5-jw04

Abstract

Cytochrome c oxidase is the terminal oxidase in the mitochondrial electron transport chain. Its function is to catalyze the four electron reduction of O2 to water and under coupled conditions the energy released in this reaction is used in the synthesis of adenosine triphosphate. The enzyme contains four metal centers, two heme a's and two coppers, all of which are inequivalent. In this work, the structure of the metal centers and the distances between the metals has been investigated by the use of electron paramagnetic resonance (EPR) and optical spectroscopy.

Cytochrome a3 and Cua3 together form the O2 binding site. In the fully oxidized enzyme these two metals do not exhibit an EPR signal due to the strong antiferromagnetic exchange interaction between these closely associated metal ions. NO has been used as a spin probe to uncouple Cua3 from cytochrome a3. In the course of these studies it was found that the enzyme catalyzed both the oxidation and reduction of NO. The analysis of these reactions has provided a new insight into the catalytic function of cytochrome£ oxidase and the implications of these experiments on the mechanism of O2 reduction is discussed. It also was found that NO binds specifically to one conformation of the oxidized enzyme. This specificity has allowed the conformations of the oxidized enzyme to be investigated. Three conformations were found to form sequentially when the reduced enzyme was reoxidized by O2. It is proposed that these conformations differ in the nature of the ligand bridging between cytochrome a3 and Cua3; the final state being one in which a μ-oxo ligand bridges between the two metal ions.

Cua is involved in transferring electrons from cytochrome c to the O2 binding site. The structure of Cua has been of considerable interest .because this site has very unusual EPR properties. The effect of sulfhydryl binding reagents on the Cua center EPR signal has been examined. These experiments together with an analysis of the EPR parameters of the native site, indicate that Cua is best described as a Cu(I)-sulfur radical complex in the oxidized enzyme. A model for the Cua center is proposed in which a Cu(I) is ligated to two neutral histidine nitrogens and two cysteine sulfurs, one a cysteinate and the other a thiyl radical, in a near tetrahedral geometry.

The distance between the metal centers was investigated by examining the electron spin relaxation properties of the Cua center by the method of continuous saturation. The dipolar interaction of a rapidly relaxing site (either of the two hemes) can dramatically increase the relaxation rate of a slowly relaxing site (the Cua center) if the sites are in close proximity. It is concluded that cytochrome a is within 13 Å of the Cua center, and that the cytochrome a3-Cua3 site is more than 10 Å from either the Cua center or cytochrome a.

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