Abstract
The site and mechanism of dioxygen reduction in cytochrome c oxidase from bovine heart muscle have been investigated. The rate of cytochrome c2+ oxidation by O2 is shown to be affected by several factors: 1) pH, with optima at 5.65 and 6.0, 2) temperature between 0 and 29 degrees C, with E alpha = 13 kcal mol-1, 3) D2O exchange, with a reduction in rate of 50% or more at the pH optima, and 4) the addition of ethylene glycol or glycerol, which significantly lowers the rate. The extremely narrow (delta vCO approximately 4 cm-1) infrared stretch bands at approximately 1964 and approximately 1959 cm-1 for liganded CO are only slightly affected by factors 1-4 or by changes in the oxidation state of metals other than the heme alpha 3 iron. These results indicate a stable, unusually immobile O2 reduction site well-isolated from the external medium, a characteristic expected to be important for oxidase function. Precise stereochemical positioning of hydrogen donors adjacent to O2 liganded to heme alpha 3 iron can be expected in order to achieve the optimization of the time/distance relationships required for enzyme catalysis. These findings support a novel mechanism of O2 reduction via a hydroperoxide intermediate within a reaction pocket that experiences little change in conformation during the hydrogen and electron transfer steps.
Highlights
The site and mechanismof dioxygen reduction in cytochrome c oxidase from bovine heart muscle have been investigated
The extremely narrow (Auto 4 cm") infrared stretch bands at -1964 and -1959 cm" for liganded CO are only slightly affected by factors 1-4 or by changes in the oxidation state of metals other than the heme a3 iron
Infrared Spectra of Cytochrome c oxidase (CcO)(0)CO"The infrared spectrum of 12C160 bound to fully reduced CcO exhibits amajor asymmetric absorbance (Fig. 1, top panel) which can be deconvoluted into two bands of Gaussian shape (Fig. 1, middle panel) [14]
Summary
The frequencies and bandwidths of the CO infrared stretch bands are sensitive to the bonding and environment of the CO ligand These parameters cangive valuable information on the ligand site structure [13]. Previous infrared studies ofCO binding to the ferrous heme u3 of CcO at different redox levels of the enzyme give clear evidence for two CO stretch bands of Gaussian shape [14].This suggests that two conformations of different struc-. The ligand environment at heme a3 remains essentially unaffected by changes in oxidation state attheother redox active metals asdemonstrated by the Mechanism of DioxyRgeednuction by CytocOhxricodmasee similar uco and AuH values for all redox levels that bind CO [14]. The reference cell for the isotope and temperature measurements contained oxidized enzyme of identical concentration placed in a precisely matched CaFz cell lytic mechanismof 0 2 reduction toHzO
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
