X-ray absorption studies have been used to investigate the structure of the four redox centers (2Fe, 2Cu) of the terminal enzyme in the respiratory chain, cytochrome c oxidase in the resting oxidized form as well as in the functional intermediates that are freeze-trapped. Methods of x-ray fluorescence detection for these low-concentration samples together with low temperature cryostats and simultaneous optical monitoring were developed to ensure good signal-to-noise data and sample integrity. The resting oxidized form contains a sulfur bridge between the copper and iron of the active site which are separated by ~ 3.8 Å. This separation of the active site metal atoms was uniquely identified by comparison of both the iron and copper EXAFS data and iron EXAFS of the copper-depleted enzyme. In the reduced state, the CO or O2 is bound to the active site iron having a structure identical to CO or oxy hemoglobin while the sulfur remains with the active site copper. Little change in structure is observed for the other iron and copper. It is the sulfur bridged active site form that is isolated by the Yonetani and Caughy methods with ≥ 85% homogeneity but not the Hartzell-Beinert or similar methods. Another form observed in the redox cycle is also fully oxidized but lacks the sulfur bridged active site with the iron of the active site having a structure identical to that of the peroxidases. This form exhibits peroxidase as well as oxidase activity, and a stable intermediate is formed with hydrogen and ethylhydrogen peroxide in which the iron of the active site is structurally similar to that of the peroxidase intermediate. The active site copper, however, does not participate in the peroxidatic role and the structures of the other iron and copper are identical to those of the sulfur bridged resting oxidized form. Thus this unique enzyme has peroxidase activity which may serve to safeguard its main oxidase function.
ASJC Scopus subject areas
- Inorganic Chemistry